Kit for detecting mRNA expression quantity of M BCR fusion gene

A technology of fusion genes and kits, applied in the biological field, to achieve fast speed, eliminate false positives and false negatives, and good specificity

Inactive Publication Date: 2013-03-13
李艳 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Real-time fluorescent quantitative PCR (Polymerase Chain Reaction, Polymerase Chain Reaction) technology has realized the leap from qualitative to real quantitative PCR, and provided an effective detection tool for the quantitative detection of human disease gene

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  • Kit for detecting mRNA expression quantity of M BCR fusion gene
  • Kit for detecting mRNA expression quantity of M BCR fusion gene
  • Kit for detecting mRNA expression quantity of M BCR fusion gene

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1. Preparation of kit of the present invention

[0034] 1. Design of specific primers and fluorescent probes

[0035] According to the gene sequence (ABL gene sequence and BCR gene sequence are from the nucleic acid database of the National Center for Biotechnology Information in the United States, the ABL gene ID is 25, and the reference sequence number is NM 005157.4; the BCR gene ID is 613, and the reference sequence number is NG_009244. 1) Design primers and fluorescent probes specific to the above gene sequences respectively.

[0036] 2. Prepare the components of the kit according to the composition of the following kits

[0037] The kit of the present invention consists of the following:

[0038] ① RNA extraction reagent: Trizol reagent (Invitrogen, product number: 15596-026 / 100ml), add 1ml Trizol to each 1ml bone marrow tissue to quickly extract RNA from bone marrow tissue of patients with chronic myelogenous leukemia.

[0039] ② cDNA first-strand s...

Embodiment 2

[0059] Embodiment 2. detect the expression level of M BCR fusion gene mRNA with the kit prepared in embodiment 1

[0060] Take the detection results of bone marrow tissue samples from 30 patients with chronic myelogenous leukemia as an example.

[0061] The detection process of using the kit of the present invention provided in Example 1 to detect the expression of M BCR fusion gene mRNA is as follows: first, design specific primers and fluorescent probes according to the gene sequence. Secondly, obtain bone marrow tissue samples from clinical leukemia patients, quickly extract tissue RNA, and perform reverse transcription PCR to synthesize the first strand of cDNA; Standards were diluted to a copy number / mL of 1.0x10 3 , 1.0x10 4 , 1.0x10 5 and 1.0x10 6 , respectively to make a standard curve of the internal positive control sequence standard (such as Figure 1A and Figure 1B shown) and ABL gene standard standard curve (as Figure 2A and Figure 2B shown), and then pr...

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Abstract

The invention relates to a fluorescent quantitative PCR (Polymerase Chain Reaction) diagnosis kit for detecting mRNA of an M BCR fusion gene, belonging to the field of biotechnology. The kit comprises a detection primer, a fluorescent probe, a cDNA first-chain synthesis reagent, a fluorescent quantitative PCR mixed liquor, negative control and positive control, wherein the detection primer and the fluorescent probe comprise an M BCR gene primer, a reference gene ABL primer and a Taqman fluorescent probe. The kit can effectively detect the M BCR fusion gene forms, such as e14a3, e13a3, e14a2 and e13a2. The M BCR fusion gene is a gene mark of malignant clone of pluripotential hemopoietic stem cells, is a typical molecular marker of chronic granulocytic leukemia and is related to inhibition of the apoptosis of the leukemic cell. The fluorescent quantitative PCR technology with higher sensitivity and specificity is used for detecting the mRNA level of the M BCR gene, and the specificity and the sensitivity of the detection result both are improved remarkably. The kit provided by the invention provides a brand-new rapid, simple and convenient gene diagnosis technology for making a therapeutic regimen for the patients with chronic granulocytic leukemia, and predicting prognosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting the expression level of M BCR fusion gene mRNA. Background technique [0002] In addition to 90% of the blood cells of patients with chronic myelocytic leukemia (CML) being positive for Ph1 chromosome, they are often accompanied by t(9;22)(q34;q11) translocation, which is located on the long arm of chromosome 9 The abl proto-oncogene is translocated to the breakpoint concentrated region bcr on the long arm of chromosome 22, forming the MBCR fusion gene. The fusion gene is responsible for encoding the P210 protein that enhances the activity of tyrosine kinase, thereby changing the tyrosine phosphorylation level of various proteins in the cell and the function of the microfilament motor protein in the cell, resulting in abnormal signal transduction in the cell and causing the cell to lose the ability to control the surrounding environment. The responsiveness of the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 李艳童永清
Owner 李艳
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