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TaqMan-COLD-PCR method for detecting JAK2V617F mutation rate in peripheral blood

A technology of JAK2V617F mutation rate, applied in the detection field of a new type of JAK2V617F mutation rate

Inactive Publication Date: 2012-01-18
AEROSPACE CENT HOSPITAL
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AI Technical Summary

Problems solved by technology

At present, there is no literature report that the detection limit of JAK2V617F mutation rate reaches 0.1%.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0009] 1. Design probes and primers.

[0010] 2. Construct wild and mutant plasmid standards.

[0011] 3. Determine the optimal denaturation temperature for TaqMan-COLD-PCR.

[0012] 4. Concentration preparation of various reagents in the PCR reaction system.

[0013] 5. Quantification of genomic DNA concentration, preparation of wild and mutant standards.

[0014] 6. Establish a standard curve for detection of mutation rate by TaqMan-COLD-PCR, and determine the detection linear range.

[0015] 7. Detection of JAK2V617F mutation rate in peripheral blood of patients, and evaluation of detection limit and mutation rate of mutation rate.

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Abstract

The invention relates to a TaqMan-COLD-PCR method for detecting the JAK2V617F mutation rate in peripheral blood. In the method, the DNA of a peripheral blood genome is used as a detection sample, the detection is completed within one reaction system, the JAK2V617F mutation percentage is quantitatively detected, and the lower detection limit is 0.1%; and the method has the characteristics of simple operation process, good repeatability, high flux of the detection sample, short report time and the like, and is especially applicable to the detection of a small quantity of mutant clone strains. The invention aims to overcome the defects of the background art and provide a method for sensitively detecting the JAK2V617F mutation rate in the DNA of a peripheral blood genome. As the unique molecular diagnosis marker of myeloproliferative diseases, the JAK2V617F mutation has been incorporated into the diagnosis standard of myeloproliferative diseases. The invention can obviously improve the diagnosis rate of myeloproliferative diseases. Meanwhile, the invention can be used for monitoring the curative effect in the treatment process of myeloproliferative diseases.

Description

technical field [0001] The patent of the present invention relates to a new detection method for the JAK2V617F mutation rate, specifically using TaqMan / MGB probe technology, combined with the COLD-PCR gene amplification principle, and quantitatively measuring the JAK2V617F mutation rate in peripheral blood genomic DNA through a Real-Time PCR analysis system Methods. Background technique [0002] In 2005, multiple research groups simultaneously discovered that the mutation at position 1849 (G>T) of the JAK2 (Janus Kinase2) gene was associated with myeloproliferative diseases (MPDs), which can obviously lead to polycythemia vera (PV), idiopathic The occurrence and development of thrombocytosis (ET) and myelofibrosis (IMF). JAK2 belongs to the Janus kinase family, and its gene is located in zone 2, zone 4, short arm of chromosome 9 (9p23-24). The JAK2 gene mutation is located at position 1849 of the JAK2 gene, and the original guanine (G) is replaced by thymine (T), making...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 梁国威
Owner AEROSPACE CENT HOSPITAL
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