Molecular beacon mediated isothermal amplification detection method and application thereof

A molecular beacon and isothermal amplification technology, applied in the field of molecular detection, can solve the problems of difficult interpretation of nucleic acid isothermal amplification technology results and limited implementation

Inactive Publication Date: 2018-05-15
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to solve the problems of difficult interpretation and limited implementation of conventional nucleic acid isothermal amplification techniques, the first object of the present invention is to provide an isothermal amplification technique mediated by molecular beacons and combined with nucleic acid chromatography detection techniques. Enhanced detection method

Method used

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  • Molecular beacon mediated isothermal amplification detection method and application thereof
  • Molecular beacon mediated isothermal amplification detection method and application thereof
  • Molecular beacon mediated isothermal amplification detection method and application thereof

Examples

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Comparison scheme
Effect test

Embodiment 1

[0074] Example 1. Detection of various contaminating bacteria in platelets by the isothermal amplification detection method mediated by molecular beacons and combined with nucleic acid chromatography detection technology

[0075] The isothermal amplification method mediated by molecular beacons and combined with nucleic acid chromatography detection technology detects multiple contaminating bacteria in platelets (simultaneous detection), including the following steps:

[0076] 1) Use DNAMAN software to compare the genomic DNA of various common contaminating bacteria in platelets (such as Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Bacillus cereus, etc., according to The target sequence (Target) was selected from the 16S rDNA conserved region of the genomic DNA of the test object, and the final target sequence was 5'-gca taa tggcgc cga cga ccg tg-3'.

[0077] Design the nucleotide sequence of a molecular beacon with a stem-loo...

Embodiment 2

[0090] Example 2. Detection of Babesia microbiota by the isothermal amplification method mediated by molecular beacons and combined with nucleic acid chromatography detection technology

[0091] The isothermal amplification detection method mediated by molecular beacons and combined with nucleic acid chromatography detection technology to detect Babesia parvum includes the following steps:

[0092] 1) Find the genome sequence of Babesia parvum that is pathogenic to humans through research literature, HQ285838,

[0093] AY027815 is the Duncan Babesia strain, BDU16370 is the divergent Babesia strain, and AF097993 is the Theileria genus. The genome DNA sequences of the four Babesia microbes were compared using DNAMAN software, and the common Babesia microbes were selected. The part of the sequence that is different from several other sequences is used as the target sequence, and the determined target sequence is:

[0094] 5'-ggtgattcataataaa-3' (matches sequence B);

[0095] 5'...

Embodiment 3

[0115] Example 3. Preparation of an isothermal amplification detection kit mediated by molecular beacons and combined with nucleic acid chromatography detection technology

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Abstract

The invention discloses a molecular beacon mediated isothermal amplification detection method combining with a nucleic acid chromatography detection technology and application thereof. The method canbe used for detecting pathogen genomes in various biofluid media, such as various unknown contaminant bacteria in blood platelet or tiny babesia propagated through blood transfusion. A molecular beacon mediated isothermal duplication product is detected by a nucleic acid chromatography method; the possibility of the isothermal duplication technology in the direction towards instant inspection (Point-Of-Care Technology, POCT) is enhanced; a kit can be further prepared; wide application can be realized in the fields of emergency/war wound emergency rescue and treatment, inspection and quarantine, sudden infectious disease inspection and monitoring, Point-of-Care Testing and the like; the application prospects are wide.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to an isothermal amplification detection method mediated by molecular beacons combined with nucleic acid chromatography detection technology and its application in instant detection. Background technique [0002] Following the development trend of clinical testing towards high-precision analysis, molecular diagnosis, as an in vitro diagnostic method with the highest technical content, is developing rapidly around the world. In 2015 alone, the world molecular diagnosis market was valued at nearly 6 billion yuan. Molecular diagnosis refers to a diagnostic technique that uses molecular biological methods to detect changes in the structure or expression level of genetic material in the body, and its core is gene diagnosis. Conventional techniques for molecular diagnosis include polymerase chain reaction (Polymerase chain reaction, PCR), DNA sequencing, single nuc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2565/625C12Q2531/119C12Q2525/30
Inventor 詹林盛王小慧蔡瑞英周欠欠张玉龙王政军何楚琳
Owner ACADEMY OF MILITARY MEDICAL SCI
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