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Kit for quantitatively detecting W515 site mutation of MPL genes

A site mutation and kit technology, applied in the field of fluorescent quantitative PCR, to achieve the effect of eliminating false positives and false negatives, good specificity, simple and safe operation

Active Publication Date: 2014-12-10
广州市宝创生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Real-time fluorescence quantitative PCR technology has realized the leap from qualitative to real quantitative PCR, and provides an effective detection tool for the quantitative detection of human disease genes. Compared with ordinary PCR, it has enhanced specificity, improved sensitivity and rapid detection. , Reduced pollution and other characteristics, but there is no relevant report on the detection kit of W515 site mutation of MPL gene by real-time fluorescent quantitative PCR method

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  • Kit for quantitatively detecting W515 site mutation of MPL genes
  • Kit for quantitatively detecting W515 site mutation of MPL genes
  • Kit for quantitatively detecting W515 site mutation of MPL genes

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1. Preparation of kit

[0038] 1. Design of specific primers and fluorescent probes

[0039]According to the gene sequence (ABL gene sequence and MPL gene sequence are from the National Center for Biotechnology Information Nucleic Acid Database, the ABL gene ID is 25, the reference sequence number is NM_005157.4; the MPL gene ID is 4352, the reference sequence number is NG_007525 .1) Design primers and fluorescent probes specific to the above-mentioned gene sequences respectively.

[0040] 2. Prepare the components of the kit according to the composition of the following kits

[0041] The kit of the present invention consists of the following:

[0042] ① Genomic DNA extraction reagent: Use tissue genomic DNA extraction kit (Qiagen Company, product number: 69504) to rapidly extract 0.5 ml of genomic DNA from the bone marrow tissue of patients with myeloproliferative neoplasms.

[0043] ② Primers, probes and standards: including MPL gene W515L site mutation-...

Embodiment 2

[0066] Embodiment 2. detect the MPL gene W515 site mutation with the kit prepared in embodiment 1

[0067] Take the detection results of bone marrow tissue samples from 30 patients with myeloproliferative neoplasms as an example.

[0068]The detection process of using the kit of the present invention to detect the mutation of the W515L site of the MPL gene and the mutation of the W515K site of the MPL gene is as follows: firstly, specific primers and fluorescent probes are designed according to the gene sequence. Secondly, obtain bone marrow tissue samples from patients with clinical myeloproliferative neoplasms, and quickly extract tissue DNA; first prepare the fluorescent quantitative PCR reaction solution of ABL internal reference gene and internal positive control sequence, and dilute the internal positive control sequence standard and ABL standard to the copy number respectively / mL is 1.0x10 3 , 1.0x10 4 , 1.0x10 5 and 1.0x10 6 , make the internal positive control se...

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Abstract

The invention relates to a kit for quantitatively detecting the W515 site mutation of MPL genes, belonging to the field of biotechnology. The kit comprises at least one system of the W515L site mutation specific system of the MPL genes and the W515K site mutation specific system of the MPL genes, and an inner reference gene ABL system, wherein each system comprises an upstream primer, a downstream primer and a Taqman fluorescent probe. Research shows that the gain-of-function mutation rates of the W515L site mutation of the MPL genes and the W515K site mutation of the MPL genes are 10% and 3% in PMF and ET respectively, and are not found in PV. Therefore, the W515L site mutation of the MPL genes and the W515K site mutation of the MPL genes are mainly used for diagnosing the PMF and the ET. With the adoption of the fluorescent quantitative PCR (polymerase chain reaction) which is high in sensitivity and specificity for detecting the W515L site mutation of the MPL genes and the W515K site mutation of the MPL genes, the specificity and the sensitivity of the detection result are remarkably improved. Via the kit disclosed by the invention, a novel rapid, simple and convenient gene diagnosis technology is provided for prediction for myeloproliferative diseases.

Description

technical field [0001] The invention relates to the fluorescent quantitative PCR technology in the field of biotechnology, in particular to a kit for quantitatively detecting the W515 site mutation of the MPL gene. Background technique [0002] Myeloproliferative diseases (MPD) are diseases in which bone marrow tissue continues to proliferate abnormally, resulting in excess red blood cells and platelets, as well as bone marrow fibrosis. According to the revision of WHO (World Health Organization, World Health Organization) in 2008, myeloproliferative diseases were changed to myeloproliferative neoplasms (myeloproliferative, MPN). MPN includes polycythemia vera (Polycythernia Vera, PV), essential thrombocytosis (Essential Thrombocytosis, ET) and primary myelofibrosis (primary myelofibrosis, PMF), all of which are diseases of excessive proliferation of blood cells, if If not treated in time, it will eventually develop into acute myelogenous leukemia. Therefore, early and rap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 童永清李艳
Owner 广州市宝创生物技术有限公司
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