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A probe combination and kit for detecting skin cancer-related markers

A technology of markers and kits, applied in the field of molecular biology, can solve the problems of not being able to well control the cross-hybridization of probes and non-specific sequences in cells, and the difficulty of simultaneous in-situ detection of expression, so as to improve detection sensitivity and specificity. Good performance and sensitivity improvement effect

Active Publication Date: 2021-03-02
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many difficulties in the multiple parallel detection of miRNA in the current prior art.
Firstly, labeled probes for each target miRNA need to be prepared separately; secondly, it is difficult to simultaneously detect the expression of multiple target miRNAs in situ
Therefore, the currently reported detection of multiple miRNAs can only use different labeling methods, however, the use of different labeling methods cannot well control the possible cross-hybridization of probes with non-specific sequences in cells

Method used

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  • A probe combination and kit for detecting skin cancer-related markers
  • A probe combination and kit for detecting skin cancer-related markers
  • A probe combination and kit for detecting skin cancer-related markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Skin cancer-related miRNA detection kit

[0053] This embodiment provides a skin cancer-related miRNA detection kit, including capture probes and signal amplification probes. The signal amplification probes include primary signal amplification probes, secondary signal amplification probes, and tertiary signal amplification probes. probe. The above probes have the characteristics of strong specificity and high sensitivity.

[0054] 1. Capture probe

[0055] The capture probe is a probe that connects the target nucleic acid and the primary signal amplification probe. The base sequence of each capture probe is from the 5' end to the 3' end in turn the specific sequence P1 that binds to the target nucleic acid to be detected, the spacer sequence , can be with the P2 sequence, the P3 sequence is a base sequence reversely complementary to the P2 sequence; the P2 sequences for different target genes are different from each other.

[0056] The spacer is used to spa...

Embodiment 2

[0087] Example 2 A kit for detecting skin cancer-related miRNA

[0088] The invention provides a skin cancer-related miRNA detection kit, which can detect target miRNAs including: hsa-miR-375, hsa-miR-149-3p, hsa-miR-221-3p, hsa-miR-222 -3p, hsa-miR-34a-5p, hsa-miR-199a-5p, hsa-miR-137, hsa-miR-182-5p, hsa-miR-125b-5p, hsa-miR-31-5p, hsa -Expression levels of miR-21-5p or hsa-miR-365a-3p, etc. In actual detection, the corresponding P1-P8 sequences can be used to form a detection kit according to specific needs, and the detection can be realized.

[0089] The components of the detection kit in this embodiment include: capture probes, signal amplification probes and fluorescent groups, and the specific probe components are shown in Table 7.

[0090] In this example, hsa-miR-375, hsa-miR-149-3p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-34a-5p, hsa-miR-199a-5p, Twelve miRNAs including hsa-miR-137, hsa-miR-182-5p, hsa-miR-125b-5p, hsa-miR-31-5p, hsa-miR-21-5p and hsa-miR-365a-3p w...

Embodiment 3

[0094] Example 3 Using the kit in Example 2 to detect the sample

[0095] This embodiment will use the kit of embodiment 2 to detect skin cancer cells,

[0096] This embodiment takes the skin cancer cell line HS-1 as an example. Those skilled in the art can obtain related cell lines in existing products according to the name of the cell lines.

[0097] The formula of described various solutions is as follows:

[0098]

[0099]

[0100] All the probes in the corresponding list in Example 1 were used in the signal amplification probe mixture in this example.

[0101] 1. Sample pretreatment, filter the sample cells onto the filter membrane

[0102] 1. Centrifuge the skin cancer cells (HS-1) in the sample storage tube at 600×g for 5 minutes, and discard the supernatant.

[0103]2. Add 4mL PBS and 1mL fixative, vortex to mix, and let stand at room temperature for 8min.

[0104] 3. Sample filtration: Transfer the liquid in the sample storage tube to the filter, turn on the...

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Abstract

The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a probe combination and a kit for detecting skin cancer-related markers. The skin cancer-related markers provided by the present invention are selected from 12 miRNAs, and the markers are closely related to skin cancer; the probe composition provided by the present invention includes a capture probe and a signal amplification composition, and the probe combination Specificity, accuracy, and sensitivity are all good, and accurate detection of target markers can be achieved with good repeatability. The kit provided by the present invention also includes a signal amplification probe. The method provided by the present invention adopts the in situ hybridization method to increase the signal intensity through a cascade amplification system. The kit and method provided by the present invention can complete the detection of the sample within 8 hours. detection, improving the detection efficiency.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a probe combination and a kit for detecting skin cancer-related markers. Background technique [0002] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs with regulatory functions found in eukaryotes, with a size of about 20-25 nucleotides. Mature miRNAs are produced by a series of nuclease cleavage processes from longer primary transcripts, and then assembled into the RNA-induced silencing complex (RNA-induced silencing complex, RISC), which is recognized by complementary base pairing target miRNA, and guide the silencing complex to degrade the target miRNA or repress the translation of the target miRNA according to the degree of complementarity. Recent studies have shown that miRNAs are involved in a variety of regulatory pathways, including development, viral defense, hematopoietic processes, organ formation, cell proliferation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/158
Inventor 刘苏燕吴诗扬董艳
Owner SUREXAM BIO TECH
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