Kit for detecting mRNA (messenger ribonucleic acid) expression quantity of U BCR fusion gene

A technology of fusion gene and kit, applied in the field of fluorescence quantitative PCR, to achieve the effect of simple and safe operation, high degree of automation and good specificity

Active Publication Date: 2013-02-13
HUAXIN SCI & TECH PANYU CITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] 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...

Method used

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  • Kit for detecting mRNA (messenger ribonucleic acid) expression quantity of U BCR fusion gene
  • Kit for detecting mRNA (messenger ribonucleic acid) expression quantity of U BCR fusion gene
  • Kit for detecting mRNA (messenger ribonucleic acid) expression quantity of U BCR fusion gene

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Embodiment 1

[0034] Embodiment 1. Preparation of kit of the present invention

[0035] 1. Design of specific primers and fluorescent probes

[0036] 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, the ABL gene ID is 25, the reference sequence number is NM_005157.4; the BCR gene ID is 613, the reference sequence number is NG_009244 .1) Design primers and fluorescent probes specific to the above-mentioned gene sequences respectively.

[0037] 2. Prepare the components of the kit according to the composition of the following kits

[0038] The kit of the present invention consists of the following:

[0039] 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.

[0040] ② cDNA first-strand synthesis kit (RT-PC...

Embodiment 2

[0060] Embodiment 2. detect the expression level of U BCR fusion gene mRNA with the kit prepared in embodiment 1

[0061] Take the detection results of bone marrow tissue samples from 30 patients with chronic myelogenous leukemia as an example.

[0062] The detection process of using the kit of the present invention provided in Example 1 to detect the mRNA expression of U BCR fusion gene is as follows: first, design specific primers and fluorescent probes according to the gene sequence. Secondly, obtain bone marrow tissue samples from patients with clinical chronic myeloid leukemia, quickly extract tissue RNA, and perform reverse transcription PCR to synthesize the first strand of cDNA; Products and ABL standard were diluted to copy number / mL of 1.0x10 3 , 1.0x10 4 , 1.0x10 5and 1.0x10 6 , to make the internal positive control sequence standard standard curve respectively (such as Figure 1A and Figure 1B shown) and ABL standard standard curve (as Figure 2A and Figur...

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Abstract

The invention discloses a kit for detecting the mRNA (messenger ribonucleic acid) expression quantity of U BCR fusion gene, belonging to the field of biotechnology. The kit comprises a detection primer, a fluorescent probe, a cDNA (complementary deoxyribonucleic acid) first strand synthesis reagent, fluorescent quantitative PCR (polymerase chain reaction) mixed solution, a negative control and a positive control, wherein the detection primer and the fluorescent probe comprise a U BCR fusion gene primer, an internal reference gene ABL primer and a Taqman fluorescent probe. The U BCR fusion gene is mainly found in chronic neutrophilic leukaemia with Ph+, and the gene code is high in the activity of tyrosine protein kinase, so that cells perform excessive multiplication and differentiation in case of no independence on cell factors, and normal cell apoptosis is inhibited. The mRNA level of the U BCR fusion gene is detected by adopting fluorescent quantitative PCR, and the specificity and the sensitivity of the detection result are remarkably improved. Via the kit, a novel rapid, simple and convenient gene diagnosis technology is provided for diagnosis, prediction and prognosis, and chemotherapy for chronic neutrophilic leukaemia.

Description

technical field [0001] The invention relates to the fluorescent quantitative PCR technology in the field of biotechnology, in particular to a kit for detecting the expression level of U BCR fusion gene mRNA. Background technique [0002] Chronic Myelocytic Leukemia (CML) is a malignant tumor caused by abnormal proliferation of pluripotent stem cells. The real understanding of the disease comes from the discovery of the Philadelphia Chromosome (Ph). Data show that more than 90% of CML Ph is present in all cases, and this chromosome is formed by the mutual translocation of chromosome 9 and chromosome 22, that is, t(9,22) (q34:q11). The translocation makes the end of the long arm of chromosome 9 (9q34) The c-abl proto-oncogene breaks at the 5' end of the second exon, and then fuses with the 3' end of the c-BCR fusion gene at the end of the long arm of chromosome 22 (22q11) to form a bcr / abl fusion gene. Among them, the breakpoint in the BCR fusion gene of some CML patients is ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 童永清李艳
Owner HUAXIN SCI & TECH PANYU CITY
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