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Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1

A technology of fusion gene and quantitative detection, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effects of high sensitivity, reduced pollution, and accurate quantification

Inactive Publication Date: 2011-06-15
上海裕隆医学检验所股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no product on the market that uses fluorescent quantitative RT-PCR technology to detect the leukemia fusion protein gene TEL-AML1

Method used

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  • Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1
  • Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1
  • Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the preparation of kit

[0043] 1. Primer and probe design and synthesis

[0044] Use Primer Express 2.0 to design and screen probes in the conserved regions in the non-fusion region of the AML1 gene and the fusion region of the TEL and AML1 genes, and design upstream and downstream primers for the AML1 gene and the TEL-AML1 fusion gene on the basis of the selected probes . Both primers and probes were synthesized by a professional company (Shenggong), wherein the primers were purified by PAGE, and the probes were purified by HPLC. The 5' end of the probe was labeled with a FAM fluorescent group, and the 3' end was labeled with a TAMRA fluorescent group.

[0045] The amplified sequence is shown in Table 1:

[0046] Table 1. Specific probe and primer sequences

[0047] sequence name

Oligonucleotide sequence (5'-3')

Base length (bp)

AML1 probe

TCAGCCTCACCCCTCTAGCCCTAC

24

TEL-AML1 probe

AGCAGAATGCATACTTGGAATGAAT...

Embodiment 2

[0062] Embodiment 2: the use of kit

[0063] 1. Sample extraction

[0064] Use RNA extraction solution to extract total RNA from the blood of the sample to be tested

[0065] Steps:

[0066] 1) Take a 0.5ml centrifuge tube, add 150μL of RNA extraction solution, then add 100μL of the sample to be tested (EDTA anticoagulant), and mix well.

[0067] 2) Add 50 μL of chloroform, centrifuge at 13000 rpm for 15 min at 4°C.

[0068] 3) Put the centrifuged upper layer into a new centrifuge tube, add 100 μL of isopropanol, and let stand at room temperature for 10 minutes.

[0069] 4) 4°C, 13000rpm, centrifuge for 15min.

[0070] 5) Gently pour off the supernatant, place it on absorbent paper, add 300 μL, ice-precooled 75% ethanol, centrifuge at 13000 rpm at 4 °C for 10 min.

[0071] 6) Gently pour off the supernatant, place it on absorbent paper, centrifuge briefly, carefully discard the supernatant with a pipette tip, and dry at room temperature for 1-5 minutes.

[0072] 7) Add ...

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Abstract

The invention discloses a fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting a leukemia translocation ETS leukemia acute myelocytic leukemia (TEL-AML)1 fusion gene and belongs to the field of in-vitro nucleic acid diagnosis. The kit comprises a quantitative reference substance, a negative reference substance, a positive reference substance, RT-PCR enzyme, PCREnhancer, diethypyrocarbonate (DEPC) water, fluorescent PCR reaction liquid I, fluorescent PCR reaction liquid II and ribose nucleic acid (RNA) extraction liquid. The kit comprises an RT-PCR system in which a fluorescent PCR technology is taken as basis, and forward and reverse primers and a fluorescent probe aiming at AMLI and TEL-AML1, can detect the RNA of AMLI and TEL-AML1 simultaneously under the same PCR condition, detect whether TEL-AML1 gene fusion occurs in a clinical sample conveniently and quickly, and provides important basis for leukemia diagnosis, typing, clinical treatment and prognosis diagnosis, and a new train of thought for clinical treatment.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, in particular to a fluorescent RT-PCR (polymerase chain reaction) kit for detecting TEL-AML1 gene fusion of leukemia patients in clinical samples. Background technique [0002] Leukemia is a malignant disease of the hematopoietic system, commonly known as "blood cancer", and is one of the top ten malignant tumors with high incidence in China. The number of leukemia patients in my country is about 3 to 4 per 100,000 population. Leukemia has the highest incidence of malignant tumors in children, and it is increasing at a rate of at least 30,000 to 40,000 per year. According to the survey, the incidence rate of leukemia in children <10 years old in my country is 2.28 / 100,000. It can occur at any age, and the incidence rate of males is higher than that of females. The activation and abnormal expression of oncogenes are related to the occurrence of leukemia, and the breakage and tran...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 穆海东汪宁梅穆宇豪黎飒
Owner 上海裕隆医学检验所股份有限公司
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