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Kit for detecting mRNA expression level of PML-RARa fusion gene

A technology that combines genes and kits, applied in the field of fluorescent quantitative PCR, to achieve simple and safe operation, low false positives, and good specificity

Active Publication Date: 2013-02-13
广州市宝创生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with immunofluorescence, real-time fluorescent quantitative PCR (Polymerase Chain Reaction, polymerase chain reaction) has the characteristics of enhanced specificity, improved sensitivity, rapid detection, and reduced pollution. At present, there is no real-time fluorescent quantitative PCR method to detect PML-RARa fusion gene

Method used

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  • Kit for detecting mRNA expression level of PML-RARa fusion gene
  • Kit for detecting mRNA expression level of PML-RARa fusion gene
  • Kit for detecting mRNA expression level of PML-RARa fusion gene

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Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1. Preparation of kit

[0042] 1. Design of specific primers and fluorescent probes

[0043] According to the gene sequence (ABL gene sequence, PML gene sequence and RARa 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 PML gene ID is 5371, refer to The sequence number is NG_029036.1; the RARa gene ID is 5914, and the reference sequence number is NG_027701.1) respectively designed primers and fluorescent probes specific to the above gene sequences.

[0044] 2. Prepare the components of the kit according to the composition of the following kits

[0045] The kit of the present invention consists of the following:

[0046] ① RNA extraction reagent: Trizol reagent (Invitrogen, product number: 15596-026 / 100ml), add 1ml Trizol to each 1ml bone marrow tissue to rapidly extract RNA from bone marrow tissue of patients with acute promyelocyti...

Embodiment 2

[0075] Embodiment 2. detect the expression level of PML-RARa fusion gene mRNA with the kit prepared in embodiment 1

[0076] Taking the results of testing 30 cases of acute promyelocytic leukemia bone marrow tissue specimens as an example, among them, the PML-RARa fusion gene forms including PML-RARa L fusion gene, PML-RARa V fusion gene and PML-RARa S fusion gene were detected in the patients . The detection process of using the kit of the present invention to detect the mRNA expression of the PML-RARa fusion gene is as follows: firstly, specific primers and fluorescent probes are designed according to the gene sequence. Secondly, obtain bone marrow tissue samples from patients with clinical acute promyelocytic leukemia, quickly extract tissue RNA, and perform reverse transcription PCR to synthesize the first strand of cDNA; The control sequence standard and the ABL standard were diluted to a copy number / mL of 1.0x10 3 , 1.0x10 4 , 1.0x10 5 and 1.0x10 6 , to make the int...

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Abstract

The invention relates to a kit for the mRNA expression level of a PML-RARa fusion gene, and belongs to the field of biotechnology. The kit comprises a PML-RARa L fusion gene system, a PML-RARa V fusion gene system or a PML-RARa s fusion gene system and a reference gene system ABL, wherein each system comprises an upstream primer and a downstream primer and Taqman fluorescence probe. The PML-RARa fusion protein, as a variant retinoic acid receptor, has different DNA properties with a wild type RARa protein and is an intrinsic and effective repressor for retinoic acid (RA) signal which is the transcription factor of an intrinsic RARa target gene. Therefore, when the fluorescence quantitative polymerase chain reaction (PCR) method is used for detecting the mRNA expression level of the PML-RARa fusion gene, the detection result is more specific and sensible. The kit provides a novel quick and simple genetic diagnosis technology for predicting the prognosis of acute promyelocytic leukemia and determining chemotherapy regimens.

Description

technical field [0001] The invention relates to fluorescence quantitative PCR technology in the field of biotechnology, in particular to a kit for detecting the mRNA expression of PML-RARa fusion gene. Background technique [0002] Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), accounting for about 10% to 15% of adult AML patients, and patients are often younger. Most of them are between 30 and 38 years old, and those under 10 years old are relatively rare. According to statistics, the incidence of APL in China is about 10% higher than that in Western countries, accounting for 18.7% of AML, and the incidence in some areas such as the Northeast Oilfield is as high as 20% to 30%. Therefore, there are age, race and geographical differences in APL. [0003] In the past ten years, due to the application of all-trans retinoic acid and arsenic trioxide, the initial induction remission rate of APL can reach more than 90% by double induction therap...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 童永清李艳
Owner 广州市宝创生物技术有限公司
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