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Colorectal cancer related microRNA detection kit

A detection kit and technology for colorectal cancer, applied in the field of molecular biology, can solve the problems of inability to control the cross-hybridization of probes and non-specific sequences in cells, and the difficulty of simultaneous in situ detection of expression, etc., to achieve practicality, Improve the fluorescence signal intensity, good specificity effect

Active Publication Date: 2020-06-02
广州益善医学检验所有限公司
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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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  • Colorectal cancer related microRNA detection kit

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0030] Example 1 Colorectal cancer-related miRNA detection kit

[0031] This embodiment provides a colorectal cancer-related miRNA detection kit, including a capture probe and a signal amplification probe, the signal amplification probe includes a primary signal amplification probe, a secondary signal amplification probe, and a tertiary signal amplification probe. probe. The above probes have the characteristics of strong specificity and high sensitivity.

[0032] 1. Capture probe

[0033] 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 , the P2 sequence that can be complementary to the P3 sequence of the primary signal amplification probe, and the P2 sequences for different target genes are different from each other.

[0034] The spac...

Embodiment 2

[0064] Example 2 A detection kit for detecting colorectal cancer-related miRNA

[0065] The invention provides a colorectal cancer-related miRNA detection kit, which can detect targets hsa-miR-21-5p, hsa-miR-602, hsa-miR-143-3p, hsa-miR-145-5p, One of hsa-miR-20a-5p, hsa-miR-181b-5p, hsa-miR-203a-3p, hsa-miR-27a-3p, hsa-miR-17-3p and hsa-miR-92a-3p In actual detection, the expression levels of any combination of one or more kinds can be detected by using the corresponding P1-P8 sequences to form a detection kit according to specific needs.

[0066] 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.

[0067] Table 7 Detection kits for specific target genes (table numbers are SEQ ID NO.)

[0068]

Embodiment 3

[0069] Example 3 Using the kit in Example 2 to detect the sample

[0070] In this example, the kit of Example 2 will be used to detect colorectal cancer cells.

[0071] The source of colorectal cancer cells is: colorectal cancer cell line HS-1, those skilled in the art can obtain related cell lines in existing products according to the name of the cell line.

[0072] The formula of described various solutions is as follows:

[0073]

[0074]

[0075] All the probes in the corresponding list in Example 2 were used in the signal amplification probe mixture in this example.

[0076] 1. Sample pretreatment, filter CTCs onto the filter membrane

[0077] 1. Preserve the blood sample in the sample preservation tube with preservation solution, centrifuge at 600×g for 5 minutes, and discard the supernatant.

[0078] 2. Add 4mL PBS and 1mL fixative, vortex to mix, and let stand at room temperature for 8min.

[0079] 3. Sample filtration: transfer the liquid in the sample stora...

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Abstract

The invention relates to a detection kit for colorectal cancer related microRNAs. The detection kit comprises at least one primary signal amplification probe, at least one secondary signal amplification probe, at least one tertiary signal amplification probe, and a capture probe, aiming at each kind of to-be-detected colorectal cancer related microRNA, wherein the P1 sequence of the capture probe for the colorectal cancer related microRNA is selected from SEQ ID NO.1 to SEQ ID NO.10. The capture probes and the signal amplification probes selected by the detection kit can be subjected to hybridization reaction under the homogeneous reaction condition, and no nonspecific binding exists among the probes; the designed probes are good in specificity and high in signal-to-noise ratio during detection. Meanwhile, due to the combined use of the various probes, the identifying kit and a detection method form a system with perfect detection effects.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a microRNA detection kit related to colorectal cancer. 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 and apoptosis, fa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6841C12Q1/682
CPCC12Q1/682C12Q1/6841C12Q2525/207C12Q2537/125C12Q2563/107
Inventor 刘苏燕吴诗扬董艳
Owner 广州益善医学检验所有限公司
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