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Reagent kit for detecting bone marrow proliferative diseases MPLW515K mutation, special-purpose primer and probe thereof

A technology of bone marrow proliferation and kits, applied to the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.

Inactive Publication Date: 2009-04-08
PEOPLES HOSPITAL PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are currently no suitable primers and specific TaqMan-MGB fluorescent probes for the detection of MPLW515K mutations on genomic DNA.

Method used

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  • Reagent kit for detecting bone marrow proliferative diseases MPLW515K mutation, special-purpose primer and probe thereof
  • Reagent kit for detecting bone marrow proliferative diseases MPLW515K mutation, special-purpose primer and probe thereof
  • Reagent kit for detecting bone marrow proliferative diseases MPLW515K mutation, special-purpose primer and probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Design of primers and TaqMan-MGB probes for detection of myeloproliferative disease MPLW515K mutation

[0041] A pair of PCR primers were designed with Primer Express Software 2.0 software based on the MPL kinase gene (GeneLoc map region of chromosome 1, 41,576,062-45,592,722bp, GeneLoc location for GC01P043576) and its reverse complement sequence, and at the same time, based on the wild-type MPL kinase gene and MPL kinase MPLW515K Two TaqMan-MGB probes were designed based on the reverse complementary sequence of the mutated gene. The 5' ends of the two TaqMan-MGB probes were respectively labeled with reporter fluorescent groups HEX and FAM, and the 3' ends were labeled with a non-luminescent quencher group. , and connected to DPI3, the sequences of primers and TaqMan-MGB probes are shown in Table 1:

[0042] Table 1 The primers and probe sequences for detecting MPLW515K mutation

[0043]

[0044] The SEQ ID NO.1 in the sequence listing consists of 21 bas...

Embodiment 2

[0046] Example 2. Detection of MPLW515K mutation in myeloproliferative disease

[0047] 1. Detection of MPLW515K mutation in myeloproliferative disease using primers designed according to the MPL genome gene and TaqMan-MGB probe

[0048] Use primers and TaqMan-MGB probes designed according to the MPL genome gene in Example 1 to MPDs patients, including ET, MF, unclassified MPDs, and bone marrow or peripheral blood samples (from People's Hospital) such as CML to carry out myeloproliferative For the detection of the disease MPLW515K mutation, the isolated bone marrow (NDM) of a normal donor is used as a control. The specific method is as follows:

[0049] 1) Extraction of genomic DNA from the sample to be tested

[0050] Genomic DNA was extracted from bone marrow samples of MPDs patients and normal bone marrow transplantation donors (see Table 1) under sterile conditions according to the DNA zol kit (purchased from Gibco, USA) and referring to the kit instructions. The method w...

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Abstract

The invention discloses a kit and a special primer and probe thereof which are used for detecting the MPLW515K mutation of myeloid proliferation diseases. The kit provided by the invention comprises a primer and a TaqMan-MGB probe; wherein, the nucleotide sequence of the primer is shown by SEQ ID NO:1 and SEQ ID NO:2 in a sequence table; the nucleotide sequence of the TaqMan-MGB probe is shown by SEQ ID NO: 3 and SEQ ID NO:4 in the sequence table. The kit provided by the invention can be used for detecting the MPLW515K mutation in clinic and scientific research, which not only provides a fast, reliable and accurate new way for diagnosing MPDs, but also can furthermore provide the exact ratio of the mutation genes of MPLW515K and the genes of wild MPL, thus providing proof for curative effect observation and the dynamic observation of minimal residual diseases.

Description

technical field [0001] The invention relates to a kit for detecting the MPLW515K mutation of a myeloproliferative disease and special primers and probes thereof. Background technique [0002] Myeloproliferative disorders (MPDs) are a group of proliferative diseases of hematopoietic stem cell tumors. On the basis of the general proliferation of bone marrow cells, a series of cells is particularly prominent, showing continuous excessive proliferation. Clinically, it can be divided into 4 types according to the main cell series of hyperplasia: ① Those with erythroid hyperplasia are mainly called polycythemia vera (PV); ② Those with granulocytic hyperplasia are called chronic granulocytic chronic myelogenous leukemia (CML); ③ the main hyperplasia of megakaryocytes is called essential thrombocythemia (essential thrombocythemia, ET); ④ the main proliferation of fibroblasts is called primary myelofibrosis Idiopathic myelofibrosis (IMF). Among them, 95% of CML patients have BCR / AB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64C12N15/11
Inventor 阮国瑞江滨陈珊珊黄晓军李金兰牛继红李玲娣
Owner PEOPLES HOSPITAL PEKING UNIV
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