Detection probe and detection method for nucleic acid aim sequence

A detection method and detection probe technology, applied in the field of molecular biology, can solve the problems such as the failure of large-scale application of isothermal amplification, difficulty in optimizing reaction conditions, and complex reaction systems, eliminating background interference, and being easy to popularize and apply. , the effect of simple reaction system

Active Publication Date: 2008-07-09
GUANGDONG HECIN SCI INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] ①The realization of these methods requires the cooperation of various enzymes, the reaction system is complex, and it is not easy to optimize the reaction conditions, and most of the enzymes or reagents used in these methods are expensive, such as: helicase in the HAD reaction, Q- Qβ replicase in β reaction, Φ29 DNA polymerase in RCA reaction, etc.
[0013] ③ The RCA reaction is the most sensitive among all isothermal amplifications, but its reaction process is complica

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  • Detection probe and detection method for nucleic acid aim sequence
  • Detection probe and detection method for nucleic acid aim sequence
  • Detection probe and detection method for nucleic acid aim sequence

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Embodiment 1

[0060] Example 1: The technical solution of the present invention is aimed at the detection of H5N1 RNA

[0061] The purpose is to demonstrate the feasibility, sensitivity and specificity of the scheme of the present invention for RNA target gene detection.

[0062] The detection process of this method is as follows image 3 As shown, the "target gene sequence" is a region of the HA gene of the H5N1 RNA virus, and a single-stranded DNA "reporter probe" (RP) is designed based on the target sequence. The 3' part of the RP is labeled with biotin, and the 5' part is labeled with SMD (sulfa-methoxine), and cross-linked with magnetic beads. Under the constant temperature reaction condition of 55°C, the RP binds to the "target gene sequence". .Under the action of BstNBI enzyme, RP is cut into two shorter fragments, which are unstable in combination with the target gene, and detached from it, the new probe can bind to the target sequence, repeat the above reaction, and finally form a...

Embodiment 2

[0076] Example 2: In the scheme of the present invention, the first method of probe modification is used to label SMD.

[0077] The first method of probe modification is to modify biotin at the 3' end of the probe, and modify small molecules, polypeptides, proteins and other substances with succinamide ester or amino structure at the 5' end; such as Figure 8 As shown, the labeling principle is to first phosphorylate the 5' end of the DNA probe and then add succinamide ester to esterify it to form a succinamide ester structure, which can then react with the amino group of SMD.

Embodiment 3

[0078] Example 3: In the protocol of the present invention, the first method of probe modification is used to label SM2.

[0079] The first method of probe modification is to modify biotin at the 3' end of the probe, and modify small molecules, polypeptides, proteins and other substances with succinamide ester or amino structure at the 5' end; such as Figure 9 As shown, the labeling principle is to first phosphorylate the 5' end of the DNA probe and then add succinamide ester to esterify it to form a succinamide ester structure, which can then react with the amino group of SM2.

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Abstract

The invention relates to a process of applying immune chromatography test paper to detect the target sequence of ribonucleotide. A detecting probe comprises identification sites of nicked endonuclease, the 3' portion of the probe is marked with biotin, and the 5'portion of the probe is marked with SMD (sulfametoxydiazine). The probe forms an integral reaction probe through incubating with magnetic beads which are marked with streptavidin. The probe is nicked into two sections by enzyme sites on the nicked endonuclease identification probe after the hybridization of the integral probe and the target sequence of ribonucleotide, which reduces the stability of the combination between the integral probe and the target sequence and separates with target sequence. The target sequence can be hybridized with another integral probe and be nicked by nick endonuclease. Therefore, a magnification mode of detection signals is formed in the reaction system. The section of the probe with magnetic beads is absorbed to deposit, and the section of the probe with SMD presents on a free state in solution after reaction solution being acted with magnetic field. The probe with SMD can be detected in detecting solution through using the immune chromatography test paper so as to reach the effect of detecting the target sequence. The process is simple, fast and economical, which is suitable to detect the target sequence of ribonucleotide in large-scale field screening.

Description

Technical field: [0001] The invention relates to the field of molecular biology, in particular to a method for detecting nucleic acid target sequences by using immunochromatographic test paper. More specifically, it is a method involving the detection of target sequences using restriction endonucleases, modified oligonucleotide probes and immunochromatographic test strips. Background technique: [0002] Fluorescent RT-PCR technology was developed in the late 1990s. This technology puts fluorescein-labeled probes and primers together and reacts in a fluorescent PCR instrument. The computer monitors the entire reaction in real time to avoid cross-contamination. It has improved the detection sensitivity and has been successfully used in the detection of human hepatitis C virus, HIV, etc., but this kind of RT-PCR-based detection method requires an expensive PCR machine for the reaction, which takes a long time and generally requires The response time of 1-2 hours is not conduci...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 彭涛高文娟曾令文李翔
Owner GUANGDONG HECIN SCI INC
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