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Nucleic acid signal amplifying detection kit

A detection kit and signal amplification technology, applied in the field of molecular biology, can solve problems such as unreliable detection, and achieve the effects of avoiding false positives, improving detection signals, and improving sensitivity

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

[0005] However, RNAscope alone still cannot reliably detect some low-copy genes historically in formalin-fixed, paraffin-embedded (FFPE) tissue sections where RNA is significantly degraded
In addition, individual RNA molecules cannot be visualized at 40× magnification using current RNAscope® technology

Method used

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  • Nucleic acid signal amplifying detection kit
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  • Nucleic acid signal amplifying detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] In this embodiment, a signal amplification detection system with high sensitivity is designed, and the signal amplification components of the first-level signal amplification probe, the second-level signal amplification probe, the third-level signal amplification probe and the capture probe will be described in detail .

[0041] 1) Level 1 signal amplification probe

[0042] The signal amplification component includes one or more primary signal amplification probes, and each primary signal amplification probe is sequenced from the 5' end to the 3' end: P4 sequence, spacer sequence, and target to be detected Nucleic acid-binding P3 sequence, the P3 sequence realizes the cascade amplification of the target signal through the combination with the capture probe.

[0043] The signal amplification probes designed by the present invention, except for the specific probes defined below in the case of reverse complementary complete matching, the sequences of P3, P4, P5, P6, P7, ...

Embodiment 2

[0071] Embodiment 2 A kind of detection kit for detecting nucleic acid

[0072] The invention provides a nucleic acid detection kit, which can detect the mRNA expression levels of target genes EGFR, KIT, B2M, etc., specifically:

[0073] 1) capture probe

[0074] Design capture probes for the mRNA of target genes such as EGFR, KIT, B2M, etc., the capture probe connects the mRNA of the target gene with the primary signal amplification probe, and the base sequence of each capture probe is from the 5' end to the 3' end In order: the specific sequence P1 capable of complementary pairing with the target gene mRNA to be detected, the spacer sequence, and the P2 sequence capable of complementary pairing with the P3 sequence of the primary signal amplification probe, the P3 sequence contains one or more The base sequence of the reverse complementary sequence;

[0075] In this embodiment, 5 capture probes are designed for each marker gene respectively, and the P2 sequence of the capt...

Embodiment 3

[0086] Example 3 Using the kit in Example 2 to detect the sample

[0087] In this example, the kits in Example 2 will be used to detect samples from different cancer cells. For details, see The following table , those skilled in the art can obtain related cell lines in existing products according to the name of the cell line. In this embodiment, the uniform cell preservation solution of each cancer cell line was taken, and the same volume of the cell preservation solution was taken as a test sample for the following experiments.

[0088] sample number cancer type Cell line name 1～5 lung cancer NCI-H1975 6～10 prostate cancer PC3 11～15 stomach cancer Ktao III 16～20 breast cancer MCF-7

[0089] The formula of described various solutions is as follows:

[0090]

[0091] The signal amplification probe mixture in this embodiment all uses the corresponding column of embodiment 2 in the table all probes.

[0092] 1. Sample pre...

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Abstract

The invention relates to a nucleic acid signal amplifying detection kit. The detection kit mainly comprises at least one first-stage signal amplifying probe and at least one second-stage signal amplifying probe or a third-stage signal amplifying probe aiming at each target gene, or mainly comprises at least one first-stage signal amplifying probe and at least one second-stage signal amplifying probe or a third-stage signal amplifying probe and a capture probe aiming at each target gene. The detection kit is designed by adopting a novel in-situ hybridization method, and fluorescent signal strength is improved through a signal amplifying system. The detection process can be completed within 8h, and a single-copied nucleic acid hybridization probe is combined with the corresponding fluorescent probe through the signal amplifying system, so that detection sensitivity of RNA in-situ hybridization is improved remarkably.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a nucleic acid signal amplification detection kit. Background technique [0002] In situ hybridization (In situ hybridization, ISH) is to use the complementary base sequence between the single strands of nucleic acid molecules to combine radioactive or non-radioactive exogenous nucleic acids (i.e. probes) with tissues, cells or chromosomes to be tested. Complementary pairing of DNA or RNA, combined into a specific nucleic acid hybrid molecule, through a certain detection method to display the position of the nucleic acid to be tested on the tissue, cell or chromosome. The DNA or RNA to be tested can be endogenous DNA, messenger RNA (mRNA), microRNA (miRNA), viral sequences or bacterial sequences. The sensitivity of this technology, that is, the threshold level of detection can reach 10-20 copies of mRNA per cell. [0003] Fluorescein label...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q2563/107C12Q2525/197
Inventor 吴诗扬许嘉森刘苏燕
Owner SUREXAM BIO TECH
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