Kit for detecting minimal residual diseases (MRD)

Abstract

The invention provides a kit for detecting lymphoid blood cancers such as T/B leukemia, lymphoma and myeloma minimal residual diseases based on research and development of high-throughput sequencing.The kit comprises multiplex-PCR primer sets for detecting IGH(VDJ), IGH(DJ), IGK, IGL, TCRbeta, TCRgamma, TCRdelta, BCL1/IGH and BCL2/IGH and with UMB (Unique Molecular Barcode, single-molecular labels), House Keeping gene PCR primer pairs, Multiplex PCR Mix(2X), internal reference DNA, Nuclease-Free Water, Elution Buffer, and primers and label sequences required for high-throughput sequencing. Bythe adoption of the kit, newly-mutant cancer cells can be found while the cancer cells are detected, the high-throughput sequencing technology is applied and combined with bioinformatic analysis, forthe detection sensitivity of MRD, one cancer cell can be at least detected in 1 million cells, that is to say, the probability is 10<-6>(0.0001%), the purpose of detecting the minimal residual diseases through quantitative analysis of the number of the cancer cells can be achieved, and sequence reading errors generated in the sequence mutation and library sequencing process due to base group mismatch during PCR amplification can be corrected.

Description

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

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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