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Primer probe method for detecting relative expression levels of TBLR1-RAR alpha and PRKAR1A-RAR alpha fusion genes

A technology for relative expression and fusion genes, applied in the field of primer probes for detecting the relative expression of TBLR1-RARα and PRKAR1A-RARα fusion genes, can solve the problems of poor specificity and high cost, and achieve simple operation, prognosis prediction and prevention The effect of clinical recurrence

Inactive Publication Date: 2018-01-12
杭州艾迪康医学检验中心有限公司
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  • Summary
  • 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; and the cost of the double-probe hybridization method is relatively expensive.

Method used

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  • Primer probe method for detecting relative expression levels of TBLR1-RAR alpha and PRKAR1A-RAR alpha fusion genes
  • Primer probe method for detecting relative expression levels of TBLR1-RAR alpha and PRKAR1A-RAR alpha fusion genes
  • Primer probe method for detecting relative expression levels of TBLR1-RAR alpha and PRKAR1A-RAR alpha fusion genes

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Experimental program
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Effect test

Embodiment 1

[0049] The invention is a method for assisting clinical diagnosis of acute myeloid leukemia and formulation of an individualized treatment plan. Mainly include the following reagents: red blood cell lysate, TRIzol, chloroform, absolute ethanol, ReverTra Ace qPCR RTKit (TOYOBO company).

[0050] Detection system PCR reaction solution: ReverTra Ace qPCR RT Kit (TOYOBO); THUNDERBIRDProbe qPCR Mix (2×), ABL internal reference gene, TBLR1-RARα, PRKAR1A-RARα target gene primers and probes are all 10 μM;

[0051] The primers and probes for detecting the internal reference gene ABL and the target gene TBLR1-RARα and PRKAR1A-RARα fusion genes are respectively:

[0052]

[0053] Positive control substance: solution containing TBLR1-RARα or PRKAR1A-RARα genome;

[0054] Negative control substance: solution without TBLR1-RARα and PRKAR1A-RARα genomes.

Embodiment 2

[0056] The operation process of the inventive method:

[0057] (1) Extraction of total RNA in 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 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 the...

Embodiment 3

[0067] The nucleic acid detection method of the present invention was used to detect samples from healthy people for physical examination, and 12 healthy physical examination samples were taken, and the genome was extracted, reagents were prepared and tested according to the method described in Example 2.

[0068] 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, internal reference gene / target gene. A 96-well fluorescent PCR instrument can detect 12 samples at the same time, each sample is repeated twice, one positive control, one negative control, and the detection time is only 100 minutes. The abl of all samples in the 12 screening samples had a line, but none of the samples of TBLR1-RARα and PRKAR1A-RARα had a line. The results are shown in Table 1

[0069] Table 1 TBLR1-RARα, PRKAR1A-RARα mRNA expression levels in 12 healthy physical examination samples

[0070]

[0071...

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Abstract

The invention discloses a method for detecting relative expression levels of TBLR1-RAR alpha and PRKAR1A-RAR alpha fusion genes by using a fluorescent quantitative PCR technology. The method uses a primer and a probe with specificity. The method can be used for detecting expression levels of the fusion genes such as TBLR1-RAR alpha, PRKAR1A-RAR alpha and PRKAR1A-RAR alpha inside acute promyelocytic leukemia (APL) patients. The method can be used as one of evidences for selection and formulation of APL individual treatment schemes, and is significant for adjusting the treatment schemes, evaluating treatment effect, predicting prognosis and preventing clinical recurrence.

Description

technical field [0001] This patent relates to a gene detection method for clinical testing. The probe method real-time fluorescent PCR technology can be used to detect the expression of TBLR1-RARα and PRKAR1A-RARα fusion genes in human acute promyelocytic leukemia, which can effectively save detection time and improve detection accuracy. Background technique [0002] Acute promyelocytic leukemia (APL) is a special subtype of acute myeloid leukemia (AML), accounting for about 17%-25.3% of AML. APL patients are mainly young and middle-aged, and the incidence is obviously age-related. The disease is dangerous and progresses rapidly. Due to disseminated intravascular coagulation and hyperfibrinolysis, there are often severe bleeding in clinical practice, and the early mortality rate is high. APL is a type of leukemia that responds better to differentiation-inducing therapy, which is related to the chromatin changes induced by the expression of retinoic acid receptor (RARα) fusi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12Q1/686
Inventor 牛林梅吴鹏飞王淑一
Owner 杭州艾迪康医学检验中心有限公司
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