Lung cancer related microRNA detection kit

A detection kit and technology for lung cancer, applied in the field of molecular biology, can solve the problems of not being able 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 increased fluorescence signal intensity, Improved detection sensitivity and high signal-to-noise ratio

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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Lung cancer related miRNA detection kit

[0030] This embodiment provides a lung cancer-related miRNA detection kit, including capture probes and signal amplification probes, signal amplification probes include primary signal amplification probes, secondary signal amplification probes, and tertiary signal amplification probes. Needle. The above probes have the characteristics of strong specificity and high sensitivity.

[0031] 1. Capture probe

[0032] 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 paired with the P3 of the primary signal amplification probe, and the P2 sequences for different target genes are different from each other.

[0033] The spacer is used to space ...

Embodiment 2

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

[0065] The present invention provides a lung cancer-related miRNA detection kit, which can detect the expression level of one or more of the following target miRNAs individually or simultaneously: hsa-miR-486-5p, hsa-miR-29a-5p , hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a-3p, hsa-miR-500a-5p, hsa-miR-424-5p, hsa-miR-10b-5p, hsa -mir-34a, hsa-miR-141-3p, hsa-miR-155-5p, hsa-miR-17-3p, hsa-miR-21-5p, hsa-miR-106a-5p, hsa-miR-146a -5p, hsa-miR-155-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa-miR-203a-3p, hsa-miR-205-5p, hsa-miR-212-3p As well as hsa-miR-214-3p, in the actual detection, according to specific needs, the corresponding P1-P8 sequences can be used to form a detection kit to realize the detection.

[0066] During detection, this embodiment uses hsa-miR-486-5p, hsa-miR-29a-5p, hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a-3p, hsa-miR -500a-5p, hsa-miR-424-5p, hsa-miR-10b-5p, hsa-mir-34a, hsa-miR-1...

Embodiment 3

[0071] Example 3 Using the kit in Example 2 to detect the sample

[0072] In this example, the kit of Example 2 will be used to detect lung cancer cells.

[0073] The source of lung cancer cells is: lung cancer cell line NCI-H1975. Those skilled in the art can obtain related cell lines in existing products according to the name of the cell line.

[0074] The formula of described various solutions is as follows:

[0075]

[0076]

[0077] All the probes in the corresponding list in Example 2 were used in the signal amplification probe mixture in this example.

[0078] 1. Sample pretreatment, filter CTCs onto the filter membrane

[0079] 1. Preserve the blood sample in the sample preservation tube with preservation solution, centrifuge at 600×g for 5 minutes, and discard the supernatant.

[0080] 2. Add 4mL PBS and 1mL fixative, vortex to mix, and let stand at room temperature for 8min.

[0081] 3. Sample filtration: Transfer the liquid in the sample storage tube to th...

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Abstract

The invention relates to a lung cancer related mciroRNA detection kit. The lung cancer related mciroRNA detection kit comprises: at least one first-grade signal amplification probe aiming at each type of lung cancer related mciroRNA to be detected, at least one second-grade signal amplification probe, at least one third-grade signal amplification probe and a capturing probe; and a P1 sequence of the capturing probe of the lung cancer related mciroRNA is selected from SEQ ID NO.1 to SEQ ID NO.21. The capturing probe and the signal amplification probes, selected by the invention, can be subjected to hybridization reaction under uniform reaction conditions, and all the probes are not in non-specifical combination; and the designed probes have good specificity and high signal-to-noise ratio. Meanwhile, a plurality of types of probes are combined so that a system with a complete detection effect is formed by utilizing an identification kit and a detection method.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a lung cancer-related microRNA detection kit. 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, fat metabol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/16C12Q2600/178
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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