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Primer, probe, composition and method for screening and identifying Ph-like ALL related fusion genes by using fluorescent PCR technology

A fusion gene, RCSD1-ABL1 technology, applied in the field of life sciences and biology, can solve the problems of high sensitivity, multiple PCR reaction systems, and analytical sensitivity that cannot meet MRD

Pending Publication Date: 2020-12-29
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

AI Technical Summary

Problems solved by technology

The detection is expensive and time-consuming, and it is not suitable for rapid screening of clinical large-scale samples
[0005] However, the method of multiple RT-PCR combined with electrophoresis takes a long time, the process is cumbersome, easy to pollute, and the judgment of the result is more subjective.
Moreover, there are many PCR reaction systems and high cost, and it is not suitable for high-throughput sample detection. It can only perform qualitative detection and cannot meet the needs of high sensitivity and specificity at the same time.
[0006] Furthermore, the analytical sensitivity of FISH detection method and multiplex RT-PCR cannot meet the needs of MRD (Minimal Residua Disease, minimal residual disease) detection

Method used

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  • Primer, probe, composition and method for screening and identifying Ph-like ALL related fusion genes by using fluorescent PCR technology
  • Primer, probe, composition and method for screening and identifying Ph-like ALL related fusion genes by using fluorescent PCR technology
  • Primer, probe, composition and method for screening and identifying Ph-like ALL related fusion genes by using fluorescent PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0208]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 inversion. Centrifuge at 4000 rpm for 3 minutes, aspirate and discard the supernatant, add red blood cell lysate to wash once to obtain the desired cells; add 1ml Total RNA Isolation Reagent, repeatedly pipetting until there are no obvious cell clumps, add 200μl of chloroform, vortex and mix for 30 seconds, and keep on ice. Set for 10min. Centrifuge at 14,000 rpm for 10 min at 4°C. Use a pipette to transfer 450 μl of the supernatant to another centrifuge tube, add an equal volume of pre-chilled isopropanol, invert and mix, and then let it stand on ice for 10 min. Centrifuge at 14,000 rpm for 10 min at 4°C. Then use 75% ethanol and absolute ethanol to wash and centrifuge once. Dry at room temperature for 5 minutes, add 50μl DEPC-H2O to dissolve.

Embodiment 2

[0210]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 and mix well, pre-denature at 70℃ for 5min; quench on ice for 1min and add 5* RTbuffer 4ul (ReverTra Ace qPCR RT Kit), Enzyme Mix 1ul (ReverTra Ace qPCR RT Kit), plus 7ul DEPC-H20 to a total volume of 20ul. After reacting at 37°C for 60 minutes and then inactivating at 98°C for 5 minutes, the result is the cDNA of the sample to be tested.

Embodiment 3

[0212]Multiple fluorescent PCR screening: configure screening reagents according to the materials and dosage shown in Table 1. The screening reagent contains a total of 8 reaction tubes: one tube from the first group to the eighth group; ABL is the internal control test in the eighth group, used to judge whether the RNA extraction quality meets the requirements. Add 2ul each of the cDNA obtained in Example 2. The detection was carried out according to the following procedure: 95°C pre-denaturation for 60s; 95°C for 10s, 58°C for 50s, a total of 40 cycles; fluorescence was collected at 58°C. The result is asFigure 6 -A: Screening showed positive FAM fluorescence in the first group.

[0213]Table 1

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[0217]

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Abstract

The invention relates to a primer, a probe, a composition and a method for screening and identifying Ph-like ALL related fusion genes by using a fluorescent PCR technology. The total number of the screened and identified related fusion genes is 35. The primer, the probe, the detection combination mode and the detection method are convenient, economical, fast, high in specificity, high in sensitivity, large in flux and suitable for clinical combination of bone marrow examination, chromosome karyotype analysis, immune typing and other results of large-batch samples to comprehensively evaluate occurrence, development and prognosis of diseases, and a basis is provided for use of targeted drugs. Meanwhile, the MRD is monitored in a targeted manner, and the recurrence risk is predicted.

Description

Technical field[0001]The present invention belongs to the field of life science and biotechnology, and relates to a primer, probe, composition and method for screening Ph-like (BCR-ABL1-like) acute lymphoblastic leukemia (ALL) related fusion genes by using multiple fluorescent PCR technology .[0002]technical background[0003]Ph-like ALL is a type of acute B lymphocytic leukemia (B-ALL) whose gene expression profile is similar to that of Ph (or BCR-ABL1 fusion gene) positive ALL; it accounts for about 10-30% of adult ALL patients, and in infants and young children About 15% of ALL patients. Ph-like ALL belongs to the poor prognosis category in the prognostic stratification of B-ALL, and its treatment options are different from those of B-ALL with good prognosis. In addition, at least 80% of Ph-like ALL patients have cytokine receptor or kinase-related gene activation changes; these patients are likely to be affected by ABL family tyrosine kinase inhibitors (such as dasatinib) and JAK ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107
Inventor 邹媛杜翠董越张辰
Owner 杭州艾迪康医学检验中心有限公司