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Primer combination and method for screening high-risk subtypes of acute lymphocytic leukemia

An acute lymphocyte and primer combination technology is applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, which can solve the problems of time-consuming, difficult acute lymphoblastic leukemia screening, and high missed detection rate.

Active Publication Date: 2020-12-01
SOUTHEAST UNIV
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Problems solved by technology

[0006] The purpose of the present invention is to provide a primer combination and method for screening high-risk subtypes of acute lymphoblastic leukemia. Aiming at the current problems of difficulty in screening high-risk subtypes of acute lymphoblastic leukemia, time-consuming, and high missed detection rate, the design is based on multiplex PCR. Technology, the primer combination and method for detecting 55 fusion genes and 206 mutation sites related to acute lymphoblastic leukemia, the primers involved have been optimized many times, with high specificity, and high-risk subtypes of acute lymphoblastic leukemia in clinical trials Screening has important guiding significance

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  • Primer combination and method for screening high-risk subtypes of acute lymphocytic leukemia
  • Primer combination and method for screening high-risk subtypes of acute lymphocytic leukemia
  • Primer combination and method for screening high-risk subtypes of acute lymphocytic leukemia

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Embodiment 1

[0133] A primer combination and method for screening high-risk subtypes of acute lymphoblastic leukemia, verifying the specificity and sensitivity of the primer combination, the patient is a relapsed and refractory Ph chromosome-negative B-ALL patient, FISH detection found that the patient has MLL -AF4 rearrangement, the first-generation sequencing test found that the patient had a CRLF2_p.A11A mutation.

[0134] The detection method is as follows:

[0135] 1. The leukemic cells of the patient were separated by density gradient centrifugation, and the human peripheral blood lymphocyte separation medium of Tianjin Haoyang Company was used;

[0136] 2. Extraction of nucleic acid: the extraction of RNA adopts the chloroform extraction method, and the extracted concentration and purity qualified RNA are reverse-transcribed to obtain cDNA, and the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit from TaKaRa Company is selected for DNA extraction. The QIAamp DN...

Embodiment 2

[0142] A primer combination and method for screening high-risk subtypes of acute lymphoblastic leukemia, verifying the specificity and sensitivity of the primer combination, the patient is a relapsed and refractory T-ALL patient, q-PCR detection found that the patient has SIL-TAL1 Fusion gene, the first-generation sequencing test found no abnormality in the patient.

[0143] The specific detection method is as follows:

[0144] 1. The leukemic cells of the patient were separated by density gradient centrifugation, and the human peripheral blood lymphocyte separation medium of Tianjin Haoyang Company was used;

[0145] 2. Extraction of nucleic acid: the extraction of RNA adopts the chloroform extraction method. The concentration and purity of the obtained RNA can be reverse-transcribed to obtain cDNA. It is recommended to use the PrimeScriptTM RT reagent Kit with gDNAEraser (Perfect Real Time) kit from TaKaRa Company. It is recommended to use the QIAamp DNA BloodMini Kit from ...

Embodiment 3

[0150] A primer combination and method for screening high-risk subtypes of acute lymphoblastic leukemia, which detects the effect of the primer combination on screening high-risk subtypes of acute lymphoblastic leukemia.

[0151] The patient was a relapsed and refractory Ph chromosome-negative B-ALL patient who relapsed only 4 months after induction of remission and died 1 month after relapse. Due to economic reasons, the patient did not undergo relevant molecular testing during clinical treatment.

[0152] The specific detection method is as follows:

[0153] 1. Use the density gradient centrifugation method to separate the leukemia cells from the patient. It is recommended to use the human peripheral blood lymphocyte separation medium from Tianjin Haoyang Company;

[0154]2. Extract nucleic acid. The extraction of RNA adopts chloroform extraction method. The concentration and purity of the obtained RNA can be reverse-transcribed to obtain cDNA. It is recommended to use TaKaR...

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Abstract

The invention discloses a primer combination and method for screening high-risk subtypes of acute lymphocytic leukemia. The primer combination comprises 55 pairs of Taqman probes and primers for fusion gene detection and 125 pairs of primers for detection of 206 mutation sites. According to the method, the high-risk subtype of the acute lymphocytic leukemia is screened by adopting a multiplex PCRtechnology and a high-throughput sequencing technology. The invention aims at common and latest molecular abnormalities of high-risk subtypes of acute lymphocytic leukemia, meanwhile, detection of fusion genes and gene mutation is carried out; the primer combination and method have the advantages of being comprehensive in range, high in accuracy and high in specificity, the amount of RNA and DNA needed for detection is low, the requirement can be met through one-time blood sampling, clinical operability is high, acute lymphocytic leukemia high-risk subtype related molecular abnormalities can be rapidly and sensitively detected, and clinical screening is guided.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a combination of primers and a method for screening high-risk subtypes of acute lymphoblastic leukemia. Background technique [0002] Acute Lymphoblastic Leukemia (ALL) is a type of hematological malignancy that is highly harmful to human health. According to its specific cell origin, it can be divided into B-cell Acute Lymphoblastic Leukemia (B- ALL) and T-cell acute lymphoblastic leukemia (Tcell Acute Lymphoblastic Leukemia, T-ALL), B-ALL and T-ALL account for about 80% and 20% of all ALL respectively. Traditional morphology combined with flow cytometry immunophenotyping can no longer meet the needs of ALL patients in the era of precision medicine. With the development of high-throughput sequencing technology, more and more molecular abnormalities related to ALL patients have been gradually discovered. Patients with these molecular abnormalities often have difficulty in clin...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12Q1/6806C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q1/6806C12Q2600/112C12Q2600/16C12Q2600/156C12Q2521/107C12Q2537/143C12Q2531/113C12Q2523/301C12Q2523/308C12Q2525/191
Inventor 葛峥韩旗葛芹玉訾杰
Owner SOUTHEAST UNIV
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