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CN-121975800-A - DUX4-IGH rearrangement detection primer probe set, kit and application

CN121975800ACN 121975800 ACN121975800 ACN 121975800ACN-121975800-A

Abstract

The invention provides a DUX4-IGH rearrangement detection primer probe set, a kit and application, wherein an upstream primer is designed on the upstream of a DUX4 transcript, 33 downstream primers are designed according to IGH cleavage positions, a universal primer is designed on the upstream of the DUX4, and a universal DUX4 probe is designed on the downstream nearby of the primers, so that the primers have small interference among the probes and small influence of non-specific amplification, are suitable for the specific detection of a plurality of DUX4-IGH rearrangement sites, solve the problem of difficult rearrangement detection in the prior art, can quantitatively detect the DUX4-IGH fusion transcript and an ABL1 reference gene in a digital PCR instrument through RT-PCR, provide basis for auxiliary diagnosis, and have remarkable application prospect.

Inventors

  • LIU HUICHAO
  • DENG SHUTING
  • ZHANG LULU

Assignees

  • 武汉希诺医学检验实验室有限公司

Dates

Publication Date
20260505
Application Date
20260205

Claims (10)

  1. The DUX4-IGH rearrangement detection primer probe set is characterized by comprising a first primer probe set, wherein the first primer probe set comprises a downstream primer with a nucleotide sequence shown as SEQ ID NO. 1-33, an upstream primer with a nucleotide sequence shown as SEQ ID NO. 34 and a probe with a nucleotide sequence shown as SEQ ID NO. 35.
  2. 2. The primer probe set of claim 1, further comprising a second primer probe set for detecting a reference gene.
  3. 3. The primer probe set of claim 2, wherein the second primer probe set comprises primers with nucleotide sequences shown in SEQ ID NOS.36-37 and probes with nucleotide sequences shown in SEQ ID NO. 38.
  4. 4. The primer probe set of claim 3, wherein any of the probes is modified with a fluorophore, and wherein different probes modify the fluorophore.
  5. 5. Use of a primer-probe set according to any one of claims 1 to 4 for the preparation of a DUX4-IGH rearrangement detection product or a DUX4-IGH fusion ratio detection product.
  6. The DUX4-IGH rearrangement detection kit is characterized by comprising a first primer probe set and a second primer probe set, wherein the first primer probe set is the DUX4-IGH rearrangement detection primer probe set according to claim 1, the nucleotide sequence of a primer in the second primer probe set is shown as SEQ ID NO. 36-37, and the nucleotide sequence of a probe is shown as SEQ ID NO. 38.
  7. 7. The test kit of claim 6, wherein the upstream primer concentration is 5uM; and/or, the concentration of any of the downstream primers is 5uM; And/or the concentration of any of the probes is 2.5 uM.
  8. 8. The test kit of claim 6, further comprising at least one of an RNA extraction reagent, a reverse transcription reagent, and a PCR amplification reagent.
  9. 9. A method for detecting a DUX4-IGH fusion gene for non-diagnostic purposes, characterized by using the primer-probe set according to any one of claims 1 to 4 or the detection kit according to any one of claims 6 to 8, comprising the steps of: S1, extracting RNA from a sample, and performing reverse transcription by using a nucleotide sequence shown as SEQ ID NO. 1-33 to obtain cDNA; s2, using cDNA as a template, mixing a nucleotide sequence shown as SEQ ID NO. 1-34 and SEQ ID NO. 35 with ddPCR Supermix, and amplifying to prepare ddPCR mixed solution; S3, preparing PCR micro-reaction liquid drops by using the ddPCR mixed solution and amplifying; S4, collecting fluorescent signals of the products after the PCR amplification reaction through a digital PCR instrument, and judging or calculating fusion gene detection results according to the fluorescent signals.
  10. 10. The method of claim 9, wherein the sample is a bone marrow or peripheral blood RNA sample.

Description

DUX4-IGH rearrangement detection primer probe set, kit and application Technical Field The invention relates to the technical field of detection kits, in particular to a DUX4-IGH rearrangement detection primer probe set, a kit and application. Background 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, with thousands of copies on the genome, and a homologous polymorphic repeat on chromosome 10. DUX4 rearrangements are found in 4% -7% of B cell acute lymphoblastic leukemia (B-ALL), mainly in children and adolescents. 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. The current common method for detecting gene rearrangement is G-banding chromosome karyotyping and FISH probe. However, for the detection of DUX4 rearrangement, none of the above-mentioned conventional detection methods detects it well for the following reasons: 1. For karyotyping, DUX4 rearrangements are cytogenetically cryptic and are more difficult to detect because of the smaller repetitive sequence inserted into the IGH site, or in the case of reverse translocation, both DUX4 and IGH are located in the subtelomere region; 2. The difficulty of FISH detection is mainly related to the fact that the reproducibility of ① D4Z4 arrays means that probes bound to this location will bind to multiple sites on 4q and 10q, which may cross-react with other highly homologous regions. ② In IGH and DUX4, the sequence and position of the breakpoint varies greatly, making the design of the separated fluorescent probe difficult. To date, detection of DUX4 rearrangement subtypes has been almost entirely dependent on NGS. Whole Genome Sequencing (WGS) has proven to be effective in detecting DUX4 rearrangements, including determining breakpoint positions, but is rarely used due to its high cost, and is not suitable as a diagnostic technique, the most widely used way in the literature is RNA-Seq detection, but the current RNA-Seq detection costs are still far higher than conventional detection methods. RT-qPCR is often used to detect various fusion genes, but DUX4-IGH rearrangements are diverse in cleavage sites at the IGH end and distributed near 1M regions, spaced farther apart from each other, and cannot be efficiently amplified by one or several primers. Disclosure of Invention The invention provides a DUX4-IGH rearrangement detection primer probe set, which is used for DUX4-IGH rearrangement detection, solves the problem of difficult detection in the prior art, and provides a basis for clinical auxiliary diagnosis. The technical scheme of the invention is realized as follows: In a first aspect of the present invention, there is provided a DUX4-IGH rearrangement detection primer probe set comprising a first primer probe set comprising a downstream primer having a nucleotide sequence as shown in SEQ ID NOS.1-33, an upstream primer having a nucleotide sequence as shown in SEQ ID NO. 34, and a probe having a nucleotide sequence as shown in SEQ ID NO. 35. Further, the DUX4-IGH rearrangement detection primer probe set also comprises a second primer probe set for detecting an internal reference gene. Preferably, the second primer probe group comprises primers with nucleotide sequences shown as SEQ ID NO. 36-37 and probes with nucleotide sequences shown as SEQ ID NO. 38. More preferably, any of the probes is modified with a fluorophore, different probes modifying the fluorophore. In a second aspect, the present invention provides an application of the primer probe set according to the first aspect in preparing a DUX4-IGH rearrangement detection product or a DUX4-IGH fusion ratio detection product. The third aspect of the invention provides a DUX4-IGH rearrangement detection kit, which comprises a first primer probe set and a second primer probe set, wherein the first primer probe set is the DUX4-IGH rearrangement detection primer probe set according to the first aspect, the nucleotide sequence of a primer in the second primer probe set is shown as SEQ ID NO. 36-37, and the nucleotide sequence of a probe is shown as SEQ ID NO. 38. Further, the concentration of the upstream primer is 5uM, and/or the concentration of any of the downstream primers is 5uM, and/or the concentration of any of the probes is 2.5 uM. Further, the detection kit further comprises at least one of an RNA extractio