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

A marker and kit technology, applied in the field of molecular biology, can solve the problems of difficulty in simultaneous in situ detection of expression, inability to control the cross-hybridization of probes and non-specific sequences in cells, etc., so as to improve detection sensitivity and specificity. Good performance and improved sensitivity

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0053] This embodiment provides an esophageal 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.

[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 spa...

Embodiment 2

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

[0088] The present invention provides a detection kit for esophageal cancer-related miRNA, which can detect target miRNA 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-25-3p, hsa-miR-373-3p, hsa-miR-16-5p, hsa-miR-208a-3p, hsa-miR-518b, hsa-miR-138-5p, 12 species including hsa-miR-145-5p, hsa-miR-296-5p, hsa-miR-21-5p, hsa-miR-223-3p, hsa-miR-192-5p...

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 esophageal cancer cells,

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

[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 esophageal cancer cells (TE-1) in the sample storage tube horizontally 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 t...

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Abstract

The invention belongs to the field of molecular biology, and relates to medicine and biotechnology, in particular to a probe composition for detecting esophagus cancer related markers and a kit of the probe composition. According to the probe composition, the esophagus cancer related markers are selected from 12 miRNAs and closely related to an esophagus cancer. The probe composition comprises a capture probe and a signal amplification composition and is good in specificity, accuracy, sensitivity and repeatability, and target markers can be accurately detected. The kit comprises a signal amplification probe. The invention provides a method which is an in-situ hybridization method, according to the method, signal strength is improved by the aid of a cascade amplification system, samples can be detected in 8h by the aid of the kit and the method, and detection efficiency is improved.

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 markers related to esophageal 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 prol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6841C12Q1/682C12N15/11
CPCC12Q1/682C12Q1/6841C12Q1/6886C12Q2600/158C12Q2600/178C12Q2525/207C12Q2565/519C12Q2563/107
Inventor 刘苏燕吴诗扬董艳
Owner SUREXAM BIO TECH
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