Method, primers and probe for detecting relative expression quantity of 11q23/MLL fusion genes

A fusion gene and MLL-AF4 technology, applied in the field of life sciences and biology, can solve the problems that are not suitable for the detection of rare and unknown abnormalities, the inability to interpret small translocations, and the high requirements of the experimental environment, so as to improve the accuracy and detection efficiency , comprehensive diagnostic guidance, good pollution control effect

Active Publication Date: 2015-06-17
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, 1) The result of traditional chromosome banding technology is intuitive, but it requires good metaphase and chromosome morphology. For duplication, deletion or inversion of small chromosome fragments, microtranslocations cannot be interpreted, and tandem duplication dup11q23 cannot be detected the exception
2) Although fluorescence in situ hybridization (FISH) has high detection sensitivity, it can only detect common chromosomal abnormalities, and is not suitable for rare and unknown abnormalities.
3) Multiplex nested RT-PCR is the most widely used method for detecting various fusion genes today. It can detect a variety of common MLL fusion genes on a large scale, but can only provide qualitative or rough quantitative results (semi-quantitative) , and the sensitivity is limited by the methodology, and there is a possibility of missed detection for the detection of small residues. More importantly, nested RT-PCR has high requirements for the experimental environment, which is prone to serious cross-contamination, resulting in false positive results
Among them, SYBR Green I, due to the use of unsaturated dyes, can produce non-specific binding to any DNA double helix structure, and its specificity must be judged by observing the melting curve, so the specificity is not ideal; the double-probe hybridization method uses two Probes, although very specific, are prohibitively expensive

Method used

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  • Method, primers and probe for detecting relative expression quantity of 11q23/MLL fusion genes
  • Method, primers and probe for detecting relative expression quantity of 11q23/MLL fusion genes
  • Method, primers and probe for detecting relative expression quantity of 11q23/MLL fusion genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] It is used to assist the clinical application of 11q23 / MLL series fusion genes MLL-AF4, MLL-AF6, MLL-AF9 and MLL in patients with ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia) and MDS (myelodysplastic syndrome) - A kit for detecting the relative expression level of ENL4mRNA, including:

[0076] Red blood cell lysate;

[0077] Trizol;

[0078] Chloroform;

[0079] Anhydrous ethanol;

[0080] Detection system PCR reaction solution: ReverTra Ace qPCR RT Kit (TOYOBO); THNDERBIRD Probe qPCR Mix (2×).

[0081] Primer and probe combinations are divided into MLL-AF4\MLL-AF6\MLL-AF9\MLL-ENL. in:

[0082]Screening group for detection of 4 fusion types of the target gene MLL-AF4: 0.8uM for the upstream and downstream primers, 0.4uM for the probe. The upstream primers are MLL-F1 and MLL-F2, the downstream primers are AF4-R1 and AF4-R2, and the probe follows the upstream primers, that is, the combination of P1 and MLL-F1, and the combination of P2 and MLL-F2...

Embodiment 2

[0118] Detection operation process:

[0119] (1) Extract tissue RNA from blood: Add 1ml of erythrocyte lysate to a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulated blood and mix well. Let stand at room temperature for 10 minutes; centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 0.5ml red blood cell lysate again, centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 1ml Trizol to the cells, and pipette repeatedly until the precipitation is complete Dissolve, let stand at room temperature for 5 minutes; add 0.2ml chloroform, shake evenly; centrifuge at 14000rpm at 4°C for 10 minutes, absorb the supernatant layer and transfer to another new centrifuge tube; add an equal volume of isopropanol, mix well up and down, and let stand at room temperature for 10 minutes Centrifuge at 14000rpm at 4°C for 10min, discard the supernatant, add 1ml of 75% ethanol, wash the tube wall upside dow...

Embodiment 3

[0139] Take 20 samples each of ALL, AML, and MDS patients submitted for inspection, extract tissue RNA according to the method described in Example 2, and reverse-transcribe the extracted tissue RNA into cDNA, prepare and detect the PCR reaction solution of the detection system.

[0140] Each sample was reverse-transcribed into cDNA, and 2 μl of cDNA sample was added to the detection system PCR reaction solution. At the same time, make positive, negative, blank control, internal reference gene / target gene standard curve each. Each sample has 2 replicates, 1 positive control, 1 negative control and 1 blank control. The detection time is 100 minutes.

[0141] Experimental result of the present invention and △ △ CT The results of the method were compared to determine the accuracy of the sample detection. The results are shown in Table 1, Table 2 and Table 3:

[0142] Table 1: Test results of 20 ALL patients

[0143]

[0144] Table 2: Test results of 20 AML patients

[01...

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Abstract

The invention discloses a method for detecting the relative expression quantity of 4 fusion genes belonging to 11q23 / MLL series, and primers and a probe for detecting the relative expression quantity of 4 fusion genes of 16 fusion types in total belonging to 11q23 / MLL series. Through test, the primers and the probe are good in specificity, high in sensitivity and simple and convenient to operate, the data which is obtained on the basis of double-standard curve method of house-keeping gene and target gene by utilizing a real-time fluorescence quantification PCR method of the Taqman probe can be used for detecting the relative expression level of the fusion genes belonging to 11q23 / MLL series in patients with ALL (Acute Lymphoblastic Leukemia), AML (Acute Myeloblastic Leukemia) and MDS (Myelodysplastic Syndrome), the high-risk groups can be accurately screened, the detection time can be effectively saved, and the detection precision can be enhanced.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular a primer, a probe and a detection method for detecting the relative expression levels of 16 fusion types in total of 4 fusion genes of 11q23 / MLL series. Background technique [0002] The MLL (mixed lineage leukemia, MLL) gene is located on the long arm of chromosome 11, region 2, band 3 (11q23), and is one of the genes that are often involved in hematologic malignancies. an important factor. So far, many partner genes on chromosome 11q23 have been identified, forming more than 70 fusion genes, at least 50 of which have been cloned at the molecular level. Recent studies have shown that 11q23 / MLL acute leukemia (11q23 / MLL acute leukemia, 11q23 / MLL AL) has different partner genes, leukemia cell types, patient age and treatment strategies, and the prognosis is very different, and these patients have different effects on conventional chemotherapy. Good, the three-year surv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/158C12Q2561/101C12Q2545/114
Inventor 周晓犊徐建成王淑一孙翠莲
Owner FUZHOU ADICON CLINICAL LAB INC
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