Nucleic acid signal amplification 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 sensitivity, and high signal-to-noise ratio

Active Publication Date: 2020-06-09
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 amplification detection kit
  • Nucleic acid signal amplification detection kit
  • Nucleic acid signal amplification detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0041] 1) Primary 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: the P4 sequence, the spacer arm sequence, and the target to be detected. Nucleic acid-bound P3 sequence, the P3 sequence realizes the cascade amplification of the target signal by binding to the capture probe.

[0043] The signal amplification probes designed in the present invention, except for the situation that the reverse complementation of the specific probes defined below is completely matched, the P3, P4, P5, P6, P7, P8...

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] Capture probes are designed for the mRNAs of target genes such as EGFR, KIT, B2M, etc., the capture probes are connected to the target gene mRNA and the primary signal amplification probe, and the base sequence of each capture probe is from the 5' end to the 3' end. The sequence is as follows: a specific sequence P1, a spacer arm sequence capable of complementary pairing with the target gene mRNA to be detected, a P2 sequence capable of complementary pairing with a primary signal amplification probe P3 sequence, and the P3 sequence contains one or more P2 sequences. The base sequence of the reverse complement of the sequence;

[0075] In this example, each marker gene is designed with 5 capture probes, the P2 sequenc...

Embodiment 3

[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, as shown in the table below. Those skilled in the art can obtain relevant cell lines in existing products according to the cell line names. In this example, the cell preservation solution of each cancer cell line was uniform, and the same volume of cell preservation solution was respectively taken as the detection sample, which was used 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 all the probes in the corresponding list in Example 2.

[0092] 1. Sample pretreatment, filter cancer...

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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, and in particular relates to a nucleic acid signal amplification detection kit. Background technique [0002] In situ hybridization (ISH) is the use of complementary base sequences between single strands of nucleic acid molecules to combine radioactive or non-radioactive exogenous nucleic acids (ie probes) with tissues, cells or chromosomes to be tested. The complementary pairing of DNA or RNA, combined into a specific nucleic acid hybrid molecule, is a technology that displays the position of the nucleic acid to be tested on the tissue, cell or chromosome by a certain detection method. The DNA or RNA to be tested can be endogenous DNA, messenger RNA (mRNA), microRNA (miRNA), viral or bacterial sequences. The sensitivity of this technique is the detection threshold level of 10-20 copies of mRNA per cell. [0003] Fluorescein labeling of in situ hybridizati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/682
CPCC12Q1/682C12Q2563/107C12Q2525/197
Inventor 吴诗扬许嘉森刘苏燕
Owner SUREXAM BIO TECH
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