Unlock instant, AI-driven research and patent intelligence for your innovation.

Kit used for detecting E2A-PBX fusion gene relative expression quantity

A relative expression level and fusion gene technology, applied in the field of genetic detection kits, can solve the problems of high cost and low specificity, and achieve the effect of simple operation, low detection cost and good specificity

Inactive Publication Date: 2013-06-05
南京艾迪康医学检验所有限公司
View PDF3 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, because SYBR GreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman method, and its specificity must be judged by observing the melting curve; and the cost of the double-probe hybridization method is relatively expensive.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Kit used for detecting E2A-PBX fusion gene relative expression quantity
  • Kit used for detecting E2A-PBX fusion gene relative expression quantity
  • Kit used for detecting E2A-PBX fusion gene relative expression quantity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The kit for detecting the relative expression of E2A-PBX fusion gene of the present invention comprises:

[0026] Red blood cell lysate;

[0027] TRIzol;

[0028] Chloroform;

[0029] Anhydrous ethanol;

[0030] Detection system PCR reaction solution: ReverTraAce qPCR RT Kit (TOYOBO); THNDERBIRD ProbeqPCR Mix (2×) E2A-PBX upstream and downstream primers 0.8uM, E2A-PBX probe 0.4uM; abl upstream and downstream primers 0.8uM, abl-probe (probe) 0.4uM;

[0031] Where: E2A-PBX-F: GGCAGCCACCCCCGAGGACG

[0032] E2A-PBX-R: TCTGTGGGTTCCTCCTCCTGG

[0033] E2A-PBX-Probe: FAM-CCTCCCCAGCCAGCCAGGCACCCT-TAMRA

[0034] abl-F: GCCGTGAAGACCTTGAAGGAG

[0035] abl-R: ATGATATAGAACGGGGGCTC

[0036] abl-Probe: FAM-ACCTGGTGCAGCTCCTTGGG-TAMRA.

[0037] Positive control substance: solution containing E2A-PBX genome; negative control substance: solution without E2A-PBX genome.

Embodiment 2

[0039] The using method of kit of the present invention:

[0040] (1) Extract tissue RNA from blood: Add 1ml of erythrocyte lysate to a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulated blood and mix well. Let stand at room temperature for 10 minutes; centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 0.5ml red blood cell lysate again, centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 1ml TRIzol to the cells, and pipette repeatedly until sedimentation Dissolve completely, let stand at room temperature for 5 minutes; add 0.2ml chloroform, shake evenly; centrifuge at 14000rpm at 4°C for 10 minutes, absorb the supernatant layer and transfer to another new centrifuge tube; add an equal volume of isopropanol, mix well up and down, and let stand at room temperature 10min; centrifuge at 14000rpm at 4°C for 10min, discard the supernatant, add 1ml of 75% ethanol, wash the tube wall up...

Embodiment 3

[0050] Using the nucleic acid detection kit of the present invention to detect clinical specimens

[0051] Sixty cases of acute lymphoblastic leukemia (ALL) samples were taken, and genomic RNA was extracted according to the method described in Example 2, and reagents were prepared and tested.

[0052] Add 2ul of each sample to the detection system PCR reaction solution. At the same time, make a standard curve of positive, negative, blank control, and internal reference gene / target gene. A 96-well fluorescent PCR instrument can detect 38 samples at the same time, each sample has 2 repetitions, a positive control, a negative control and a blank control. The detection time is only 100 minutes.

[0053] The experimental results are compared with the results reported by the special inspection laboratory to determine the accuracy of the sample detection. Some positive results are as follows:

[0054]

[0055] Table 1 shows the comparison between the results of this experiment...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a kit used for detecting an E2A-PBX fusion gene relative expression quantity. The kit used for detecting the E2A-PBX fusion gene relative expression quantity comprises red blood cell lysate, TRIzol, chloroform, absolute ethyl alcohol, ReverTraAceqPCRRTKit, detecting system polymerase chain reaction (PCR) reaction liquid, a positive reference substance and a negative reference substance. The kit used for detecting the E2A-PBX fusion gene relative expression quantity is characterized in that the detecting system PCR reaction liquid comprises THUNDERBIRDqPCRMIX, a forward primer E2A-PBX-F and a reverse primer E2A-PBX-R used for detecting purpose gene, a probe namely an E2A-PBX-Probe, primers abl-F and abl-R used for detecting the reference gene Abl, and a probe namely an abl-Probe. According to the kit used for detecting the E2A-PBX fusion gene relative expression quantity, an E2A-PBX fusion gene expression level in a body of an acute lymphoblastic leukemia patient can be detected. Detection time can be effectively saved, and detection precision is improved.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to a gene detection kit, which can detect the expression level of E2A-PBX fusion gene in patients with acute lymphoblastic leukemia by using probe real-time fluorescent quantitative PCR technology, which can effectively save Detection time, improve detection accuracy. Background technique [0002] Acute Lymphoblastic Leukemia (ALL) is a type of malignant blood disease caused by the clonal proliferation, accumulation, and tissue infiltration of malignant lymphocytes. Among them, the E2A / PBX1 fusion gene formed by t(1;19)(q23;pl3) translocation is found in about 5% of children, especially in B-cell ALL, which accounts for 20-25%. t(1;19) fuses E2A at 19p13 with PBX1 at 1q23. The complete E2A as a transcription factor includes the transcriptional activation domain and the HLH domain, and E2A can promote transcriptional activation. In the past, children with ALL who were...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 周晓犊徐建成王淑一孙翠莲
Owner 南京艾迪康医学检验所有限公司