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 section

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
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Example Embodiment

[0039] Example 1

[0040] In this embodiment, a set of high-sensitivity signal amplification detection system 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) Primary signal amplification probe

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

[0043] The signal amplification probe designed in the present invention, except for the specific probe reverse complementation and perfect match as defined below, the P3, P4, P5, P6, P7, P8 sequence does not have a hairpin...

Example Embodiment

[0071] Example 2 A detection kit for detecting nucleic acid

[0072] The present invention provides a nucleic acid detection kit, which can detect the mRNA expression level 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 target gene mRNA with the first-level signal amplification probe, and the base sequence of each capture probe is from 5'end to 3'end The sequence is: a specific sequence P1, a spacer arm sequence that can complement and pair with the mRNA of the target gene to be detected, and a P2 sequence that can be complementarily paired with the P3 sequence of the primary signal amplification probe. The P3 sequence contains one or more and P2 The base sequence of the reverse complement of the sequence;

[0075] In this example, 5 capture probes were designed for each marker gene, and the P2 sequence of the capture probe was selecte...

Example Embodiment

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

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

[0088] Sample number Cancer Cell line name 1~5 Lung cancer NCI-H1975 6~10 Prostate cancer PC3 11~15 Stomach cancer KtaoIII 16~20 Breast cancer MCF-7

[0089] The formulations of the various solutions are as follows:

[0090]

[0091] The signal amplification probe mixture in this example uses the corresponding column of Example 2. In the table All probes.

[0092] 1. Sample pretreatment to filter cancer cells onto the filter membrane

[0093] 1. Use the cancer cell sample preserved in ...

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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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