A kit for detecting minimal residual disease mrd

A minimal residual disease and kit technology, applied in the biological field, can solve problems such as high requirements for experimental conditions and operating techniques, low standardization, and inability to quantify

Active Publication Date: 2022-07-19
杭州艾沐蒽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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  • A kit for detecting minimal residual disease mrd
  • A kit for detecting minimal residual disease mrd
  • A kit for detecting minimal residual disease mrd

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Obtaining the sample genome

[0069] 1. According to before and after treatment and sample specificity, it can be divided into:

[0070] 100uL, 200uL, 300uL, 400uL, 500uL of human bone marrow samples before treatment were placed in EDTA anticoagulation tubes;

[0071] 100uL, 200uL, 300uL, 400uL, 500uL, 600uL, 700uL, 800uL, 900uL, 1mL, 2mL of bone marrow samples after human treatment were placed in EDTA anticoagulation tubes;

[0072] Human peripheral blood samples of 5mL, 6mL, 7mL, 8mL, 9mL, and 10mL were placed in EDTA anticoagulation tubes.

[0073] 2. Use the red blood cell lysate to lyse the red blood cells in the sample and separate the nucleated live cells.

[0074] 3. The obtained nucleated live cells were counted and the genomic gDNA was extracted.

Embodiment 2

[0075] Example 2: Multiplex PCR Amplification and Library Construction

[0076] Using the library building kit in the kit, add multiple pairs of primers of V gene fragment and J gene fragment with UMB into the multiplex PCR reaction system, and add any three corresponding 1% input DNA template amount of known The internal reference DNA or House Keeping gene of the sequence is specifically amplified at the same time as the sample.

[0077] The primer set sequences are shown in Table 1

[0078] Table 1 Multiplex PCR primers

[0079]

[0080]

[0081]

[0082]

[0083] The primer sequence structure is as figure 2 shown, wherein the Read1 sequence is SEQ ID NO: 248, and the Read2 sequence is SEQ ID NO: 249.

[0084] The sequence of the internal reference DNA is shown in SEQ ID NOs: 128-226.

[0085] The PCR primer sequences for amplifying the House Keeping gene are shown in SEQ ID NOs: 250-253.

[0086] Multiplex PCR system, including 25μL, 50μL, the following is...

Embodiment 3

[0092] Example 3: High-throughput sequencing and bioinformatics analysis

[0093] The method of the present invention uses the Hiseq system from illumina company. Hiseq is a single-molecule cluster-based sequencing-by-synthesis technology based on a proprietary reversible termination chemical reaction principle. During sequencing, random fragments of DNA are attached to the optically transparent glass surface (Flow cell). After extension and bridge amplification, these DNA fragments form hundreds of millions of clusters (clusters) on the Flow cell. Thousands of single-molecule clusters of the same template. Then, four kinds of special deoxyribonucleotides with fluorescent groups are used to sequence the template DNA to be tested by reversibly terminated sequencing by synthesis (Sequencing by Synthesis, SBS) technology.

[0094] First, the primers and tags of the illumina high-throughput sequencer were added to both ends of the PCR1 product, and amplified at the same time (see...

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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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2531/113C12Q2535/122C12Q2537/143C12Q2545/101
Inventor 孙涛余莹莹张天骄
Owner 杭州艾沐蒽生物科技有限公司
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