Probe combination and kit for detecting myeloma-associated 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, improve the effect of detection signal

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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  • Probe combination and kit for detecting myeloma-associated markers
  • Probe combination and kit for detecting myeloma-associated markers
  • Probe combination and kit for detecting myeloma-associated markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Myeloma-related miRNA Detection Kit

[0053] This embodiment provides a myeloma-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 spacer is used to space ...

Embodiment 2

[0086] Example 2 A detection kit for myeloma-associated miRNA

[0087] The invention provides a myeloma-related miRNA detection kit, which can detect target miRNAs including: hsa-miR-34b-5p, hsa-miR-20a-5p, hsa-miR-15a-5p, hsa-miR -16-5p, hsa-miR-135b-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-34a-5p, hsa-let-7e -5p or hsa-miR-21-5p and other expression levels, in actual detection, according to specific needs, the corresponding P1-P11 sequences can be used to form a detection kit to achieve detection.

[0088] 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.

[0089] In this example, hsa-miR-34b-5p, hsa-miR-20a-5p, hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-135b-5p, hsa-miR-29a- 11 miRNAs including 3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-34a-5p, hsa-let-7e-5p or hsa-miR-21-5p were randomly divided into 3 groups, To test ...

Embodiment 3

[0093] Example 3 Using the kit in Example 2 to detect the sample

[0094] This embodiment will use the kit of embodiment 2 to detect myeloma cells,

[0095] This embodiment takes the myeloma cell line GBC-SD as an example. Those skilled in the art can obtain related cell lines in existing products according to the name of the cell line.

[0096] The formula of described various solutions is as follows:

[0097]

[0098]

[0099] All the probes in the corresponding list in Example 1 were used in the signal amplification probe mixture in this example.

[0100] 1. Sample pretreatment, filter the sample cells onto the filter membrane

[0101]1. Centrifuge the myeloma cells (GBC-SD) in the sample storage tube horizontally at 600×g for 5 minutes, and discard the supernatant.

[0102] 2. Add 4mL PBS and 1mL fixative, vortex to mix, and let stand at room temperature for 8min.

[0103] 3. Sample filtration: Transfer the liquid in the sample storage tube to the filter, turn on...

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Abstract

The present invention belongs to the field of molecular biology, and relates to medicine and biotechnology, particularly to a probe combination and a kit for detecting myeloma-associated markers, wherein the myeloma-associated markers are selected from 11 miRNAs and are closely related to myeloma, and the probe combination comprises a capture probe and a signal amplification composition, has advantages of good specificity, good accuracy, good sensitivity and good reproducibility, and can achieve the accurate detection of target markers. The kit further comprises a signal amplification probe. According to the present invention, with the kit and the method, by using the in-situ hybridization method, through the cascade amplification system, the signal intensity is increased so as to improve 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 myeloma-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) through complementary base pairing. Recognize the 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 刘苏燕吴诗扬董艳
Owner SUREXAM BIO TECH
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