Primers, probes and kit for detecting leukemia-related fusion genes

A technology of fusion gene and detection primer, which is applied in the field of genetic detection in molecular biology, can solve the problems of limited clinical application, low detection sensitivity, cumbersome and complicated operation, etc., and achieve the effect of good specificity, high sensitivity and simple operation

Inactive Publication Date: 2016-02-03
武汉海吉力生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems of expensive, cumbersome and complicated operations, limited clinical application, and low detection sensitivity in the pri

Method used

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  • Primers, probes and kit for detecting leukemia-related fusion genes
  • Primers, probes and kit for detecting leukemia-related fusion genes
  • Primers, probes and kit for detecting leukemia-related fusion genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the extraction of clinical sample RNA

[0038] In this embodiment, RNA is extracted from peripheral blood of leukemia patients and quantified as a template for PCR detection. The RneasyMinikit blood RNA extraction kit provided by Qiagen was used, and the operation was performed according to the instructions, as detailed below:

[0039] a. Add an equal volume of RLT to 0.5-1.5 mL of fresh anticoagulated whole blood sample, vortex or invert gently to mix, and place on ice for 3-5 minutes.

[0040] b. Add an equal volume of absolute ethanol at room temperature and mix well.

[0041] c. Add 700μL to the separation column, centrifuge at 10000rpm for 15s.

[0042] d. Wash the separation column with 700μL of RW1, centrifuge at 10000rpm for 15s.

[0043] e. Rinse the separation column with 500 μL washing liquid RPE, centrifuge at 10000 rpm for 15 s.

[0044] f. Rinse the separation column again with 500 μL washing liquid RPE, centrifuge at 12000 rpm for 2 min....

Embodiment 2

[0047] Example 2: The kit detects the effect of clinical samples

[0048] Leukemia-related fusion gene fluorescent PCR detection kit contains 8 splicing isomers of four common leukemia fusion genes, BCR / ABL, PML / RARα, AML1 / ETO and TEL / AML1. The detection system detects 8 fusion gene splice isoforms sequentially as follows: BCR / ABL (e1a2), BCR / ABL (e13a2), BCR / ABL (e14a2), BCR / ABL (e19a2), PML / RARαL type, PML / RARαS, AML1 / ETO, and TEL / AML1.

[0049] Table 2 Composition of fluorescent PCR detection kit for leukemia-related fusion genes

[0050]

[0051]

[0052] (Note: RT enzyme is reverse transcriptase, which is used to reverse transcribe RNA into cDNA; TaqDNase is DNA polymerase; buffer is reaction buffer; probe is TaqMan-MGB probe)

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Abstract

The invention discloses primers, probes and a kit for detecting leukemia-related fusion genes. The primers and probes are designed according to specificity of eight types of fusion genes such as BCR/ABL, PML/RAR alpha, AML1/ETO and TEL/AML1. The primers, probes and kit have high sensitivity and good specificity. The primers, probes and kit can detect 10 copies of a mutational sample. The kit utilizes a one-step real-time fluorescent quantitative reverse transcription polymerase chain reaction method to detect a sample to be detected, realizes reverse transcription and quantitative PCR in a reaction system, only needs one-step fluorescent PCR amplification, is free of a reagent and pipe cover opening in the reaction process, has detection time of 90min and is operated simply. The kit has the advantages of high sensitivity, good specificity, low price, fastness and simple operation and has a high scientific research value and a high clinical application value.

Description

technical field [0001] The invention relates to gene detection of molecular biology in the field of biotechnology, in particular to primers, probes and kits for detecting leukemia-related fusion genes. Background technique [0002] Chronic myeloid leukemia (CML) is a malignant clonal proliferative disease of the blood system that occurs in hematopoietic stem cells. 90% to 95% of the abnormal white blood cells of CML patients can be detected to contain a chromosomal translocation, which is named Philadelphia (Ph) chromosome. The formation mechanism of Ph chromosome is that a section of chromosome 9 is spliced ​​to chromosome 22, and this splicing forms an oncogenic fusion gene BCR / ABL. The protein encoded by the BCR / ABL gene has abnormal tyrosinase activity, which keeps the signal transduction pathway regulating cell replication in an active state, thereby continuously producing more abnormal white blood cells, and destroying the production of white blood cells normally cont...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156
Inventor 王秀娟李存耀段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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