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Primer, probe, composition and method for screening and identifying fusion genes related to PAX5 rearrangement by using fluorescent PCR technology

A fusion gene, PAX5-ETV6 technology, applied in the field of life sciences and biology, can solve the problems of high cost, low sensitivity, and long time consumption

Active Publication Date: 2021-02-26
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of FISH is high, and the sensitivity is not as good as that of PCR; while the method of PCR combined with electrophoresis takes a long time, the process is cumbersome, easy to pollute, and the judgment of the result is more subjective
And the more PCR reaction systems, the higher the cost, which is not suitable for high-throughput sample screening

Method used

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  • Primer, probe, composition and method for screening and identifying fusion genes related to PAX5 rearrangement by using fluorescent PCR technology
  • Primer, probe, composition and method for screening and identifying fusion genes related to PAX5 rearrangement by using fluorescent PCR technology
  • Primer, probe, composition and method for screening and identifying fusion genes related to PAX5 rearrangement by using fluorescent PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Extraction of RNA from whole blood: Add 1ml of 1× red blood cell lysate to a 1.5ml centrifuge tube, take 0.5ml of the whole blood sample to be tested, and mix by inverting. Centrifuge at 4000rpm for 3min, discard the supernatant, add erythrocyte lysate and wash once to obtain the desired cells; add 1ml Total RNA Isolation Reagent, blow repeatedly until no obvious cell clumps, add 200μl of chloroform, vortex mix for 30s, static on ice Set for 10min. Centrifuge at 14,000rpm at 4°C for 10min. Use a pipette to draw 450 μl of the supernatant and transfer it to another centrifuge tube, add an equal volume of pre-cooled isopropanol, invert and mix well, and then let stand on ice for 10 minutes. Centrifuge at 14,000rpm at 4°C for 10min. Then wash and centrifuge once with 75% ethanol and absolute ethanol respectively. Dry at room temperature for 5 min, add 50 μl DEPC-H2O to dissolve.

Embodiment 2

[0131] Reverse transcription: Take 4ul of the RNA solution in Example 1 (concentration about 200ng / ul), add 1ul Primer mix (ReverTraAce qPCR RT Kit) and 3ulDEPC-H2O, mix well, pre-denature at 70°C for 5min; add 5* after quenching on ice for 1min RTbuffer 4ul (ReverTra Ace qPCR RT Kit), Enzyme Mix 1ul (ReverTra Ace qPCR RT Kit), and add DEPC-H20 7ul to a total volume of 20ul. After reacting at 37°C for 60 minutes, it was inactivated at 98°C for 5 minutes, and the cDNA of the sample to be tested was obtained.

Embodiment 3

[0133] Multiplex fluorescent PCR screening: Configure screening reagents according to the materials and dosages shown in Table 1 below. The screening reagent contains a total of 3 reaction tubes: the first and second tubes are the screening detection group; the third tube is the ABL1 internal reference gene detection group, which is used to judge whether the quality of RNA extraction meets the requirements. 2ul of each cDNA obtained in Example 2 was added. Detection was performed according to the following procedure: pre-denaturation at 95°C for 60s; 10s at 95°C and 50s at 58°C, a total of 40 cycles; fluorescence was collected at 58°C. The result is as figure 2 Shown: Screening shows a negative result.

[0134] Table 1

[0135]

[0136]

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PUM

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Abstract

The invention relates to a primer, a probe, a composition and a method for screening and identifying fusion genes related to PAX5 rearrangement by using a fluorescent PCR technology. The screened andidentified fusion genes comprise 17 types of genes including PAX5-ETV6, PAX5-JAK2, PAX5-ELN, PAX5-FOXP1, PAX5-PML, PAX5-ZNF521, PAX5-AUTS2, PAX5-KIDINS220, PAX5-ESRRB, PAX5-NOL4L, PAX5-HIPK1, PAX5-BRD1, PAX5-POM121, PAX5-DACH1, PAX5-DACH2, PAX5-NCOR1 and PAX5-GOLGA6A. The primer, the probe, a detection combination mode and a detection method are convenient, economical, rapid, good in specificity,high in sensitivity, large in flux and suitable for comprehensive assessment of occurrence and development of diseases through clinical combination with bone marrow examination, chromosome karyotype analysis, immune typing and other results of large-batch samples; and meanwhile, MRD monitoring is conducted in a targeted mode, and the recurrence risk is predicted.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a primer, a probe, a composition and a method for screening PAX5 rearrangement-related fusion genes by using multiple fluorescent PCR technology. [0002] technical background [0003] About 47% of children and 34% of adult B-ALL patients can detect PAX5 gene abnormality, which is one of the most common genetic abnormalities in B-ALL, and the incidence of PAX5 gene rearrangement is about 14%. There are more than 20 fusion genes involved in PAX5 rearrangement, including PAX5-ETV6, PAX5-JAK2, PAX5-ENL, PAX5-PML, and PAX5-FOXP1. Cell differentiation plays an important role in the occurrence and development of B-ALL. Due to the small number of cases involving PAX5 rearrangement, there is no clear conclusion on the classification of the prognosis of B-ALL with such abnormalities, but it can still be used as a supplementary marker for ALL screening, which is hel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/158C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/101C12Q2563/107
Inventor 邹媛董越杜翠张辰
Owner FUZHOU ADICON CLINICAL LAB INC
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