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Multiplex RT-PCR (reverse transcription-polymerase chain reaction) reagent kit for detecting leukemia BCR-ABL (Abelson proto-oncogene-breakpoint cluster region)

A RT-PCR and leukemia technology, applied in the field of leukemia detection, can solve the problems of limited application, limited qualitative research, expensive instruments and reagents, etc., and achieve the effect of low sample requirements, sensitive and fast detection, and simple operation

Inactive Publication Date: 2014-03-12
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Fluorescent quantitative PCR technology has become the focus of minimal residual disease monitoring research due to its automated program operation, high sensitivity, and quantitative advantages, but there are not many qualitative studies, and the required instruments and The expensiveness of the reagent undoubtedly limits its application in the initial clinical diagnosis and typing of leukemia

Method used

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  • Multiplex RT-PCR (reverse transcription-polymerase chain reaction) reagent kit for detecting leukemia BCR-ABL (Abelson proto-oncogene-breakpoint cluster region)
  • Multiplex RT-PCR (reverse transcription-polymerase chain reaction) reagent kit for detecting leukemia BCR-ABL (Abelson proto-oncogene-breakpoint cluster region)
  • Multiplex RT-PCR (reverse transcription-polymerase chain reaction) reagent kit for detecting leukemia BCR-ABL (Abelson proto-oncogene-breakpoint cluster region)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0023] Example 1 BCR-ABL fusion gene detection kit and its use

[0024] 1 Prepare a kit including the following components: 1 tube of BCR-ABL gene PCR reaction solution (1200 μL), 1 tube of Taq enzyme (30 μL), and 1 tube of control substance (300 μL).

[0025] 2 Collection, storage and transportation of samples:

[0026] 2.1 Type of sample used: fresh blood.

[0027] 2.2 Sample collection, preservation and transportation: RNA extraction is performed immediately after fresh blood is collected with an anticoagulant tube, or white blood cells are extracted and frozen in liquid nitrogen and stored at -80°C. Samples are shipped on dry ice over long distances.

[0028] 3. Nucleic acid extraction:

[0029] It is recommended to use the MagCore Compact Nucleic Acid Extractor and operate according to the instructions of its matching whole blood RNA extraction kit (MRN-01). Finally, an RNA sample with an elution volume of 200 μL can be obtained from every 400 μL blood sample, and reve...

Embodiment 2

[0037] Example 2 Application of BCR-ABL fusion gene detection kit to detect 5 cases of clinical samples

[0038] The blood samples of 2 patients with chronic myelogenous leukemia and 2 patients with acute lymphoblastic leukemia identified by karyotype analysis and 1 healthy blood sample without BCR-ABL were selected, and the RNA was extracted immediately after reverse transcription, and the obtained reverse transcription The recorded product was detected, and the reference substance and NTC were detected at the same time.

[0039] Interpretation of test results: The no-template control substance NTC of the BCR-ABL fusion gene has no other peaks except the marker chromatographic peak (20bp, 100bp), indicating that the system is not contaminated; the control substance has three fusion forms with the BCR-ABL fusion gene The corresponding peaks of e1a2, e13a2 and e14a2 are 413bp, 215bp and 290bp, indicating that the system can effectively detect the BCR-ABL fusion gene.

[0040] ...

Embodiment 3

[0042] Example 3 Simultaneous detection of 50 clinical samples using the BCR-ABL fusion gene detection kit and the clinical gold standard karyotype analysis technique

[0043] A total of 50 leukemia blood samples were selected, among which 45 cases were identified as positive by clinical gold standard karyotype analysis, and 5 cases were identified as negative. Immediately after RNA extraction, reverse transcription was performed, and the obtained reverse transcription product was detected with a BCR-ABL fusion gene detection kit. The results are shown in Table 1.

[0044] Table 1 Comparison of two methods for detecting BCR-ABL fusion gene results

[0045]

[0046] Note: McNemar test was used for paired four-table data, χ 2 =0, P>0.05, Kappa=0.756

[0047] Table 1 shows the comparison between the results of the detection kit and the karyotype analysis. The sensitivity of the multiple RT-PCR detection of the BCR-ABL fusion gene established by this kit is 97.8% (44 / 45), and t...

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Abstract

The invention discloses a multiplex RT-PCR (reverse transcription-polymerase chain reaction) reagent kit for detecting leukemia BCR-ABL (Abelson proto-oncogene-breakpoint cluster region) and relates to leukemia detection. With the adoption of a technology of multiplex RT-PCR in combination with capillary electrophoresis, three fusion forms, namely e1a2, e13a2 and e14a2, of the BCR-ABL can be detected simultaneously by a single PCR, and the detection time and cost are effectively saved. The length of an amplification product can be judged quickly, efficiently and accurately according to a chromatogram by taking the three fusion forms, namely e1a2, e13a2 and e14a2, constructed by an overlap extension method as positive quality controls. The kit is applicable to multiple fields of qualitative detection and diagnosis, selection of auxiliary clinical medication and prognosis judgment of the different fusion forms, namely e1a2, e13a2 and e14a2, of BCR-ABL fusion genes in acute lymphoblastic leukemia patients and a few acute myeloid leukemia patients.

Description

technical field [0001] The invention relates to the detection of leukemia, in particular to a multiple RT-PCR kit for detecting leukemia BCR-ABL. Background technique [0002] Leukemia is a malignant clonal disease in the hematopoietic system. In my country, the incidence rate is about (3.0-4.0) / 100,000, and the mortality rate is 50%. In the mortality rate of malignant tumors, leukemia ranks 6th and 8th among men and women in my country (Sun Xiaodong, Chai Yihuan, Cao Youfu, etc. Investigation and Research on the Incidence Factors of Childhood Leukemia[J]. China Journal of Aerospace Medicine, 2003, 12(6) :1-3). Many studies have shown that most leukemia patients have some kind of chromosomal abnormality (translocation, inversion, deletion, etc.), which is an important reason for the development of leukemia. Chronic myeloid leukemia (chronic myeloid leukemia, abbreviated as CML) was the first to link chromosomal abnormalities with specific tumor types. The incidence of CML ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2537/143C12Q2565/125
Inventor 曾骥孟吕晓楠刘祝君
Owner XIAMEN UNIV
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