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Detection primers, probes and detection kits for human alk gene expression

A gene expression and kit technology, applied in the fields of biotechnology and medicine, can solve problems such as high requirements for operation and interpretation technology, difficulty in lung cancer cell interpretation, and high cost of FISH, so as to ensure authenticity and prevent false positive results The effect of short generation and detection time

Active Publication Date: 2020-12-15
WUHAN YZY MEDICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

1) FISH detection has high requirements for operation and interpretation techniques; 2) Patients with advanced NSCLC can only provide small biopsy tissues of about 2 mm, and it is difficult to ensure that there are more than 50 lung cancer cells in each visual field for interpretation
3) The judgment cut-off value (cut-off value) of FISH test results is also questionable. Many studies have found that a small number of ALK FISH-negative and IHC-positive patients can also benefit significantly from crizotinib treatment
4) The current cost of FISH testing is expensive
But this method has following deficiency: 1) detection process takes a long time, needs 160 minutes
2) The specificity of the system has not been verified, such as the tolerance of the system to genomic DNA and wild-type samples
3) No quality control system was introduced to monitor the quality of the samples, which may result in false negative results due to poor sample quality
4) The result interpretation method of the system is relatively complicated, and the clinical applicability is slightly poor

Method used

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  • Detection primers, probes and detection kits for human alk gene expression
  • Detection primers, probes and detection kits for human alk gene expression
  • Detection primers, probes and detection kits for human alk gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] This example is used to illustrate the preparation of the human ALK gene expression detection kit of the present invention.

[0101] Specifically include the following steps:

[0102] 1. Primer and probe synthesis:

[0103] Design and synthesize 4 sets of specific primers: SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:8; 4 specific probes SEQ ID NO:9-12, and the 5' end of the probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a NFQ-MGB non-luminescent quencher. The primers and probes were prepared into 100 μM stock solutions for storage.

[0104] 2. Prepare a quality control system: design and synthesize a pair of quality control primers for the human B2M gene, the primer pair sequences are SEQ ID NO:13 and SEQ ID NO:14; design and synthesize a quality control probe, the probe is SEQ ID NO:14 ID NO: 15. The quality control primers and probes were respectively prepared into ...

Embodiment 2

[0113] The ALK gene detection kit prepared in Example 1 was used to detect the FFPE samples of wild-type lung cancer patients to evaluate the specificity of the system; to detect the positive plasmids to evaluate the sensitivity of the system.

[0114] In this example, 20 FFPE samples of ALK-fused wild-type lung cancer patients were collected, and DNA and RNA were extracted from them respectively, wherein the DNA was directly detected with the ALK gene expression detection kit obtained in Example 1, and the RNA was reverse-transcribed into cDNA , detected with the ALK gene expression detection kit obtained in Example 1, and evaluated the specificity of the kit. At the same time, the positive plasmid was extracted, and after dilution, it was detected with the ALK gene expression detection kit obtained in Example 1 to evaluate the sensitivity of the system.

[0115] 1. Sample preparation

[0116] 1. FFPE sample DNA preparation

[0117] Use the Qiagen 56404 kit to extract DNA f...

Embodiment 3

[0176] The ALK gene detection kit prepared in Example 1 was used to detect the samples to be tested.

[0177] In this example, 200 FFPE samples of lung cancer patients were collected, RNA was extracted from them, reverse-transcribed into cDNA, and the ALK expression of the samples to be tested was detected with the ALK gene expression detection kit obtained in Example 1.

[0178] 1. RNA extraction from FFPE samples

[0179] (a) Take the above lung cancer samples, add 1mL xylene respectively, mix well, centrifuge at 13000rpm for 2 minutes at room temperature, discard the supernatant, add 1mL absolute ethanol to the precipitate, shake and mix well (remove xylene), and Centrifuge at 13000rpm for 2 minutes, discard the supernatant, open the cap of the centrifuge tube, and place it at 37°C for 10 minutes to evaporate the residual ethanol;

[0180] (b) Add 240 μL Buffer PKD and 10 μL proteinase K to the centrifuge tube, shake and mix, incubate at 56°C for 15 minutes, incubate at 80...

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Abstract

The invention relates to the field of biotechnologies and medicines and in particular discloses a detection primer, a probe and a detection kit for human ALK (Anaplastic Lymphoma Kinase) gene expression. The detection primer comprises sequences shown as SEQ ID NO: 1 to SEQ ID NO: 8; the sequence of the probe is shown as SEQ ID NO: 9 to SEQ ID NO: 12; a 5' end of the probe is modified by FAM or VIC and a 3' end of the probe is modified by NFQ-MGB. The primer and the probe, provided by the invention, can be used for determining whether ALK gene fusion exists or not through detecting expression difference of the 5' end and 3' end of an ALK gene. The kit contains the detection primer and the probe, and a quality control system and the like. The detection primer, the probe and the detection kit can be used for detecting mutation which is as low as 5 copies and can tolerate reverse transcripts of 600ng of genome DNA (Deoxyribonucleic Acid) and 5mu g of wild RNA (Ribonucleic Acid); a whole reverse transcription PCR (Polymerase Chain Reaction) and fluorescent PCR detection process can be finished within only 80min.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to the detection of human ALK gene expression. Background technique [0002] Anaplastic lymphoma kinase (ALK) was first discovered in a subtype of anaplastic large cell lymphoma (ALCL). The ALK gene is located at chromosome 2p23, contains 29 exons, and can encode a type I transmembrane protein ALK with 1620 amino acids and a molecular weight of 200KDa. ALK protein is a receptor tyrosine kinase (receptor tyrosine kinase, RTK) and a member of the RTK insulin superfamily. The complete ALK has a three-part structure, namely the extracellular region, the lipophilic transmembrane region and the intracellular tyrosine kinase, and its amino acid kinase domain consists of 254 (positions 1123 to 1376) amino acid residues. [0003] The protein expression of ALK is related to the differentiation of neuroectoderm. Normal ALK is exclusively expressed in the nervous system, such as the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2561/101C12Q2563/107C12Q2545/113
Inventor 蔡从利张喆李丽琼周鹏飞
Owner WUHAN YZY MEDICAL SCI & TECH
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