Primers, probes, compositions and methods for screening and identifying pax5 rearrangement-related fusion genes using fluorescent PCR technology

A technology of fusion gene, PAX5-ETV6, applied in the field of life science and biology, can solve the problems of subjectivity, time-consuming, easy to contaminate and so on

Active Publication Date: 2022-06-17
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The FISH method is costly and less sensitive than PCR; while the method of PCR combined with electrophoresis takes a long time, the process is cumbersome, easy to contaminate, 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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  • Primers, probes, compositions and methods for screening and identifying pax5 rearrangement-related fusion genes using fluorescent PCR technology
  • Primers, probes, compositions and methods for screening and identifying pax5 rearrangement-related fusion genes using fluorescent PCR technology
  • Primers, probes, compositions and methods for screening and identifying pax5 rearrangement-related fusion genes using fluorescent PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] 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, invert and mix. Centrifuge at 4000 rpm for 3 min, aspirate and discard the supernatant, add erythrocyte lysate and wash once to obtain the desired cells; add 1 ml of Total RNA Isolation Reagent, pipette repeatedly until there is no obvious cell clumps, add 200 μl of chloroform, vortex and mix for 30 s, and keep on ice. Set 10min. Centrifuge at 14,000 rpm for 10 min at 4°C. Transfer 450 μl of the supernatant to another centrifuge tube with a pipette, add an equal volume of pre-cooled isopropanol, invert and mix, and then let stand on ice for 10 min. Centrifuge at 14,000 rpm for 10 min at 4°C. 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

[0130] Reverse transcription: Take 4ul of RNA solution in Example 1 (concentration about 200ng / ul), add 1ul Primer mix (ReverTraAce qPCR RT Kit) and 3ul DEPC-H2O to mix, pre-denature at 70°C for 5min; quench on ice for 1min, add 5* RTbuffer 4ul (ReverTra Ace qPCR RT Kit), Enzyme Mix 1ul (ReverTra Ace qPCR RT Kit), and add DEPC-H20 7ul to the total volume of 20ul. After the reaction at 37°C for 60min, inactivation was performed at 98°C for 5min, and the result was the cDNA of the sample to be tested.

Embodiment 3

[0132] Multiplex fluorescent PCR screening: configure screening reagents according to the materials and dosages shown in Table 1 below. The screening reagent contains 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 RNA extraction quality meets the requirements. Each 2ul of the cDNA obtained in Example 2 was added. The detection was performed according to the following procedure: pre-denaturation at 95°C for 60s; 95°C for 10s, 58°C for 50s, a total of 40 cycles; fluorescence was collected at 58°C. The result is as figure 2 Shown: Screening showed negative results.

[0133] Table 1

[0134]

[0135]

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Abstract

The present invention relates to primers, probes, compositions and methods for screening and identifying fusion genes related to PAX5 rearrangement using fluorescent PCR technology. The related fusion genes screened and identified include 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 , PAX5‑GOLGA6A, a total of 17 species. The primers, probes, detection combinations and detection methods of the present invention are convenient, economical, fast, specific, high in sensitivity, and large in throughput, and are suitable for clinical combination of large batches of samples with bone marrow examination, karyotype analysis, and immunoassay. Comprehensively assess the occurrence and development of the disease based on results such as type, and at the same time carry out targeted monitoring of MRD to predict the risk of recurrence.

Description

technical field [0001] The invention belongs to the fields of life science and biotechnology, and particularly relates to a primer and a probe, a composition and a method for screening PAX5 rearrangement-related fusion genes by using multiple fluorescent PCR technology. Background technique [0002] 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. 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, etc. These translocations lead to abnormal expression of PAX5 gene, thereby blocking B lymphocytes 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 for the classification of the prognosis of B-ALL with such abnorm...

Claims

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Application Information

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Patent Type & Authority Patents(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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