Ovarian cancer-related microRNA detection kit

A detection kit and ovarian cancer technology, applied in the field of molecular biology, can solve the problems of difficult simultaneous in situ detection of expression, inability to well control the cross-hybridization of probes and non-specific sequences in cells, etc., to improve the intensity of fluorescent signals , simple design, strong specific effect

Active Publication Date: 2017-05-10
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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Examples

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Comparison scheme
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Embodiment 1

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

[0031] This embodiment provides an ovarian 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 spacer is...

Embodiment 2

[0065] Example 2 A detection kit for detecting ovarian cancer-related miRNA

[0066] The invention provides a kit for detecting ovarian cancer-related miRNA, which can detect targets hsa-miR-21-5p, hsa-miR-141-3p, hsa-miR-200a-3p, hsa-miR-200c- The expression level of one or more of 3p, hsa-miR-200b-3p, hsa-miR-203a-3p, hsa-miR-205-5p and hsa-miR-214-3p in any combination, in actual detection According to the specific needs, the corresponding P1-P8 sequences can be used to form a detection kit, and the detection can be realized. The ovarian cancer-related miRNA detection kit described in this example was randomly divided into 2 groups for detection in the following detection.

[0067] 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.

[0068] Table 7 Detection kits for specific target genes (table numbers are SEQ ID NO.)

[0069]

[00...

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 ovarian cancer cells.

[0073] The source of ovarian cancer cells is: ovarian cancer cell line SK-OV-3. Those skilled in the art can obtain related cell lines in existing products according to the name of the cell lines.

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

[0075]

[0076] All the probes in the corresponding list in Example 2 were used in the signal amplification probe mixture in this example.

[0077] 1. Sample pretreatment, filter CTCs onto the filter membrane

[0078] 1. Preserve the blood sample in the sample preservation tube with preservation solution, centrifuge at 600×g for 5 minutes, and discard the supernatant.

[0079] 2. Add 4mL PBS and 1mL fixative, vortex to mix, and let stand at room temperature for 8min.

[0080] 3. Sample filtration: Transfer the liquid in the sample storage tube to the fi...

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Abstract

The invention relates to an ovarian cancer-related microRNA detection kit. In accordance with each to-be-detected ovarian cancer-related microRNA, the detection kit comprises at least one first-grade signal amplification probe, at least one second-grade signal amplification probe and at least one third-grade signal amplification probe, as well as a capture probe, wherein a P1 sequence of the capture probe of the ovarian cancer-related microRNA is selected from SEQ ID NO.1-SEQ ID NO.8. According to the detection kit provided by the invention, the selected capture probe and signal amplification probes, a hybridization reaction can be conducted under a uniform reaction condition, and non-specific binding can be avoided between the various probes; and the designed probes are good in specificity and high in signal-to-noise ratio in detection. Meanwhile, by combining the various probes, a system, which is complete in detection effect, is formed by the identifying kit and a detection method.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to an ovarian 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 apopto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/16C12Q2600/178
Inventor 刘苏燕吴诗扬董艳
Owner SUREXAM BIO TECH
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