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Primers and method for detecting expression level of leukemia cdx2 gene

A CDX2-F and CDX2-R technology, applied in the field of primers and methods for detecting the expression level of CDX2 gene in leukemia, can solve the problems of high cost and inferior specificity, and achieve the effects of simple operation, prediction of prognosis and high precision

Active Publication Date: 2020-01-21
FUZHOU ADICON CLINICAL LAB INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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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

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  • Primers and method for detecting expression level of leukemia cdx2 gene
  • Primers and method for detecting expression level of leukemia cdx2 gene
  • Primers and method for detecting expression level of leukemia cdx2 gene

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Experimental program
Comparison scheme
Effect test

Embodiment 2

[0056] The operation process of the inventive method:

[0057] (1) Extraction of total RNA in blood: Add 1ml of erythrocyte lysate into 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 1500rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 0.5ml red blood cell lysate again, centrifuge at 1500rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 1ml TRIzol to the cells, and pipette repeatedly until the precipitation is complete Dissolve, 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 for 10 minutes Centrifuge at 14000rpm at 4°C for 10min, discard the supernatant, add 1ml of 75% ethanol, wash t...

Embodiment 3

[0067] Embodiment 3 Sensitivity detection of the present invention

[0068] Experiments were carried out with plasmid concentrations of 100, 10, and 1 copies / μL as templates, each concentration was repeated 10 times, and a blank control was set at the same time. As a result, it is found that the detection limit of the present invention is 10copies, specifically see Table 1 and figure 1 .

[0069] Table 1 Sensitivity detection result of the present invention

[0070]

[0071]

Embodiment 4

[0072] Embodiment 4 adopts detection method described in the present invention to detect healthy population sample

[0073] Take 20 cases of healthy physical examination samples to be tested, extract genomes, prepare reagents and detect according to the method described in Example 2.

[0074] Add 2 μL 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 20 samples at the same time, each sample has 2 repetitions, a positive control, a negative control and a blank control, and the detection time is only 100 minutes. The abl of all the samples in the 20 screening samples had a line, but no sample of CDX2 had a line. The results are shown in Table 2, and the detection result figure of sample 1 is shown in figure 2 .

[0075] Table 2 CDX2mRNA expression levels of 20 healthy physical examination samples ...

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Abstract

The invention discloses primers and a probe for detecting relative expression quantity of CDX2 gene and a detection method. The forward and reverse primers for amplification coverage of CDX2 gene and the probe are respectively CDX2-F, CDX2-R and CDX2-Probe. The forward and reverse primers and the probe for detecting reference gene Abl are respectively Abl-F, Abl-R and Abl-Probe. The detection reagent and the method have high precision, and results are convenient to read and discriminate. In addition, specificity is good, sensitivity is high, and operation is simple. The invention is helpful for clinical early diagnosis of leukemia and monitoring of minimal residual disease (MRD), and is of great significance for timely intervention of treatment, adjustment of therapeutic schedule, evaluation of treatment effect, prediction of prognosis and prevention of clinical relapse.

Description

technical field [0001] The invention relates to a gene detection method and molecular detection primers for clinical testing. The probe real-time fluorescent quantitative PCR technology can be used to detect the expression level of CDX2 in human leukemia, which can effectively save detection time and improve detection accuracy. Background technique [0002] Leukemia is a kind of malignant clonal disease with abnormal development of hematopoietic stem cells. With the development of my country's economy, the aggravation of environmental pollution and the change of lifestyle, the incidence of leukemia is on the rise, especially among young and middle-aged people under the age of 35. and high rates of leukemia morbidity and mortality among children. The prognosis of leukemia is generally poor, there is no good prevention and control measures, and the case fatality rate is high. At present, it is believed that minimal residual disease (MRD) is the root cause of leukemia recurrenc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/166
Inventor 牛林梅黄开新王淑一
Owner FUZHOU ADICON CLINICAL LAB INC
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