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Oligonucleotide and method for detecting expression level of NPM1-RARA fusion gene

A technology of fusing genes and oligonucleotides, applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc. problem, to achieve the effect of good specificity, high sensitivity, and reduction of operation time and steps

Pending Publication Date: 2020-12-11
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Quantification of fusion genes can detect minimal residual lesions and provide evidence for disease progression and treatment effects; the disadvantage is that due to many influencing factors, the false positive and false negative rates are high, and the design of primers also affects the detection
FISH method detection is more intuitive than real-time fluorescent quantitative PCR, but in comparison, FISH has low sensitivity and can only be qualitative

Method used

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  • Oligonucleotide and method for detecting expression level of NPM1-RARA fusion gene
  • Oligonucleotide and method for detecting expression level of NPM1-RARA fusion gene
  • Oligonucleotide and method for detecting expression level of NPM1-RARA fusion gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The nucleic acid detection method used to detect the relative expression of NPM1-RARa-1 and NPM1-RARa-2 in samples can be used to assist in the clinical diagnosis of acute myeloid leukemia and the formulation of individualized treatment plans, and can also predict the prognosis of patients . The reagents and components of the kit include:

[0049] (1) RNA extraction reagent, RNAsimple Total RNA kit (TIANGEN)

[0050] (2) RNA reverse transcription reagent, ReverTra Ace qPCR RT Kit kit (TOYOBO company).

[0051] (3) Detection system PCR reaction solution, THNDERBIRD Probe qPCR Mix (2×) (TOYOBO Company).

[0052] Use upstream and downstream primers NPM1-RARa-1-F, NPM1-RARa-2-F, RARa-R to detect the target gene, and the probe is RARa-Probe; detect the internal reference gene Abl with primers Abl-F, Abl-R, probe The needle is Abl-Probe. The specific primer and probe base sequences are as follows:

[0053] NPM1-RARa-1-F:5'-CCTACCTAAGTGCGTGCC-3';

[0054] NPM1-RARa-2-F:5...

Embodiment 2

[0062] 1. Operation procedure for extracting total RNA from peripheral blood or bone marrow:

[0063] (1) Collect 1ml of anticoagulated fresh blood or bone marrow, and register age and gender;

[0064] (2) Take fresh blood or bone marrow directly, add 3 times the volume of RZ (recommended 0.25ml blood + 0.75ml RZ), shake and mix well.

[0065] (3) Place the homogenized sample at 15-30° C. for 5 minutes to completely separate the nucleic acid-protein complex.

[0066] (4) Optional step: centrifuge at 12,000 rpm (~13,400×g) at 4°C for 5 min, take the supernatant, and transfer it to a new RNase-free centrifuge tube.

[0067] (5) Add 200 μL of chloroform, cap the tube, shake vigorously for 15 sec, and place at room temperature for 3 min.

[0068] (6) Centrifuge at 12,000rpm (~13,400×g) for 10min at 4°C. The sample will be divided into three layers: a yellow organic phase, an intermediate layer and a colorless aqueous phase. RNA is mainly in the aqueous phase, and the volume of t...

Embodiment 3

[0087] 1. Sensitivity test

[0088] For NPM1-RARa-1 and NPM1-RARa-2, 1000, 100, and 10 copies of plasmids were used as templates for experiments, and 10 tubes of each concentration were repeated for sensitivity testing. The results showed that the detection sensitivity of both NPM1-RARa-1 and NPM1-RARa-2 was 100copies / μL.

[0089] 2. Specificity test

[0090] Take 11 cases of peripheral blood samples from healthy people who submitted for examination, extract RNA according to the method described in Example 2, perform reverse transcription, prepare reagents, and perform fluorescence quantitative PCR detection.

[0091] For each sample, 2 μL of cDNA obtained by reverse transcription was added to the detection system PCR reaction solution. At the same time, make one copy of positive control, negative control and blank control, and two copies of standard curve of internal reference gene / target gene. A 96-well fluorescent PCR instrument can detect 16 samples at the same time, ea...

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Abstract

The invention provides oligonucleotide and detection method for detecting the expression level of a NPM1-RARa fusion gene in the body of a patient suffering from acute promyelocytic leukemia. The oligonucleotide comprises a primer and probe for detecting the NPM1-RARa-1 and NPM1-RARa-2 fusion genes. A real-time fluorescence PCR technology and a double-standard curve method using a detection primerand probe of a target gene and a reference gene are adopted, so that expression levels of fusion genes NPM1-RARa-1 and NPM1-RARa-2 in the body of the patient suffering from APL can be detected. The detection time can be effectively saved, and the detection precision is improved. Besides, the oligonucleotide and the detection method can be used as one of bases for selecting and formulating an APLindividualized treatment scheme, have important significance for adjusting a treatment scheme, evaluating the treatment effect, predicting prognosis and intervening treatment in time to avoid hematological recurrence, and are convenient for clinically performing individualized treatment.

Description

technical field [0001] This patent relates to the field of molecular detection and diagnosis, and specially designs oligonucleotides, kits and detection methods using probe Taqman real-time fluorescent quantitative PCR technology to detect the NPM1-RARA fusion gene. Background technique [0002] Acute promyelocytic leukemia (APL) is a special subtype M3 of AML (acutemyeloid 1eukemia), accounting for about 15% of AML. The age of onset is mainly young and middle-aged people over 20 years old. High bleeding tendency and early mortality. More than 95% of APL patients can see the translocation of chromosome 15 and chromosome 17, forming a characteristic t(15;17)(q22;q21), that is, PML-RARa fusion gene, which is a characteristic genetic marker of APL. [0003] The PML-RARa protein encoded by the PML-RARa fusion gene can form a dimer with RXRa, compete for the combination of wild-type RARa and RXRa, prevent the differentiation and maturation of granulocytes, and at the same time, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851
Inventor 牛林梅陈雪青王淑一
Owner FUZHOU ADICON CLINICAL LAB INC
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