Assay kit for testing relative expression of core-binding factor (CBF) beta/myosin 11 fusion genes

A relative expression level and fusion gene technology, applied in the field of gene detection kits, can solve the problems of high cost and poor specificity, and achieve the effects of simple operation, prognosis prediction and prevention of clinical recurrence

Inactive Publication Date: 2012-11-28
南昌艾迪康医学检验实验室有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, since 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; while the cost of the double-probe hybridization method is relatively expensive

Method used

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  • Assay kit for testing relative expression of core-binding factor (CBF) beta/myosin 11 fusion genes
  • Assay kit for testing relative expression of core-binding factor (CBF) beta/myosin 11 fusion genes
  • Assay kit for testing relative expression of core-binding factor (CBF) beta/myosin 11 fusion genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The kit for detecting the relative expression of CBFβ / MYH11 fusion gene of the present invention comprises:

[0031] Red blood cell lysate;

[0032] TRIzol;

[0033] Chloroform;

[0034] Anhydrous ethanol;

[0035] Detection system PCR reaction solution: ReverTra Ace qPCR RT Kit (TOYOBO); THNDERBIRD Probe qPCR Mix (2×), CBFβ / MYH11 upstream and downstream primers each 0.8uM, CBFβ / MYH11 probe 0.4uM; abl upstream and downstream primers each 0.8uM, abl-probe (probe) 0.4uM; where:

[0036] CBFβ / MYH11-F: GGAGGATGCATTAGCACAACAG;

[0037] CBFβ / MYH11-A-R: TCTCCTCCATCTGGGTC;

[0038] CBFβ / MYH11-D-R: ATCCCTGTGACGCTTCCAACT;

[0039] CBFβ / MYH11-E-R: GCTTATTCTTGTCTAGGTTCGCC;

[0040] CBFβ / MYH11-Probe: FAM-GAAGAGGCTCGGAGAAGGACAC-TAMRA;

[0041] abl-F: GCCGTGAAGACCTTGAAGGAG;

[0042] abl-R: ATGATATAGAACGGGGGCTC;

[0043] abl-Probe: FAM-ACCTGGTGCAGCTCCTTGGG-TAMRA;

[0044] Positive control substance: solution containing CBFβ / MYH11 genome; negative control substance: solution wi...

Embodiment 2

[0046] The using method of kit of the present invention:

[0047] (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 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, stand at room temperature Centrifuge at 14000rpm at 4°C for 10min, discard the supernatant, add 1ml of 75% ethanol, wash the tube wall upside down gently; ...

Embodiment 3

[0057] The nucleic acid detection kit of the present invention is used to detect clinical specimens.

[0058] A total of 60 anticoagulated blood samples from acute myelogenous leukemia (acute myelognous leukemia, AML) patients were collected, and genomic RNA was extracted, reagents were prepared and tested according to the method described in Example 2.

[0059] 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.

[0060] 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:

[0061]

[0062] Table...

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Abstract

The invention discloses an assay kit for testing the relative expression of core-binding factor (CBF) beta/myosin 11 fusion genes, and the kit comprises red blood cell lysis buffer, TRIzol, chloroform, absolute ethyl alcohol, ReverTraAceqPCRRTKit, testing system polymerase chain reaction (PCR) reaction solution, positive control substance and negative control substance, and is characterized in that the testing system PCR reaction solution comprises THUNDERBIRDqPCRMIX, primers of CBF beta/MYH11-F, CBF beta/MYH11-A-R, CBF beta/MYH11-D-R and CBF beta/MYH11-E-R for amplifying a target gene, probe of CBF beta/MYH11-Prob, primers of abl-F and abl-R for amplifying an internal control gene, and probe of abl-Probe. The assay kit can be used for testing the expression level of the CBF beta/myosin 11 fusion genes of a patient suffering from human acute myelognous leukemia (AML), so the test time can be effectively saved, and the test 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 adopts probe real-time fluorescent quantitative PCR technology and is used for detecting CBFβ / MYH11 fusion in human acute myeloid leukemia (acute myelognous leukemia, AML) patients At the same time, more accurate screening of high-risk groups is carried out. The kit can effectively save detection time and improve detection accuracy. Background technique [0002] Acute myelocytic leukemia is acute myeloid leukemia. Its disease progresses rapidly, and its natural course is only a few weeks to several months. At present, no real cause has been found. It is generally believed that genetics, radiation, chemical substances (such as benzene), drugs and other occupational Exposure to certain substances (such as smoke, paint, pesticides, etc.) may be related to the occurrence of AML. Clinically, acute myeloid leukemia is divided into 8 subtypes, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 徐建成周晓犊王淑一
Owner 南昌艾迪康医学检验实验室有限公司
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