A kit for detecting minimal residual disease mrd

Abstract

The invention provides a kit for detecting lymphoid blood cancers (such as T / B lineage leukemia, lymphoma, myeloma minimal residual disease) developed based on high-throughput sequencing, the kit includes detecting IGH (VDJ), IGH (DJ), IGK, IGL, TCRβ, TCRγ, TCRδ, BCL1 / IGH, BCL2 / IGH multiplex PCR primer set with UMB (Unique Molecular Barcode, single molecule tag), House Keeping gene PCR primer pair, Multiplex PCR Mix (2X), internal reference DNA, Nuclease‑Free Water, Elution Buffer, and primers and index sequences required for high-throughput sequencing. Using the kit, newly mutated cancer cells can be found while detecting cancer cells, and by applying high-throughput sequencing technology combined with bioinformatics analysis, the detection sensitivity of MRD can detect at least one cancer cell in 1 million cells , which is 10 ‑6 (0.0001%), not only can quantitatively analyze the number of cancer cells to achieve the purpose of minimal residual disease detection, but also can correct sequence mutations caused by base mismatches generated by PCR amplification and sequence reading errors generated during library sequencing.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a kit for detecting minimal residual disease MRD. Background technique [0002] Lymphatic hematological cancers mainly include T / B lymphocytic leukemia, lymphoma and multiple myeloma, while minimal residual disease (MRD) refers to the remission of leukemia / lymphoma / myeloma patients in the body after induction therapy. A state in which a small number of cancer cells remain, which may eventually lead to disease recurrence. Leukemia can be divided into lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, is a kind of malignant clonal diseases of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in bone marrow and other hematopoietic tissues due to mechanisms such as uncontrolled proliferation, impaired differentiation, and blocked apoptosis, and infiltrate other non-hematopoietic tissues and organs, while inhibiting normal hematopoietic function. Lymph...

AI Technical Summary

Problems solved by technology

The most commonly used method is flow cytometry (Flow Cytometry) based on molecular immunology, but its sensitivity can only reach 10 -4 order of magnitude (0.01%), and the accuracy of this method for judging MRD results depends largely on the experimental conditions of each laboratory and the personal experience of the operator, the degree of standardization is low, and some studies have shown that during chemotherapy due to The influence of chemotherapy drugs changes the immune phenotype of cancer cells, the phenomenon of "immune drift", which can affect the reliability and accuracy of MRD detection results

The method of polymerase chain reaction (PCR) cannot be quantified, only the gene rearrangement of IG / TCR can be seen

Fusion gene real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) detection method can only be applied to acute leukemia with abnormal fusion gene, and cannot indicate the source of leukemia cells

The application of allelic oligonucleotide hybridization method (AS0-PCR) is more common in Europe. This method needs to customize a set of primers according to each patient, which is prone to non-specific amplification, and requires high experimental conditions and operating techniques. , time-consuming and labor-intensive, and mutations in the nucleic acid of cancer cells will cause false negatives in MRD detection

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