Multiplex nucleic acid visualization detection method and kit based on solid phase rolling circle amplification and particle aggregation

A rolling circle amplification and multiple nucleic acid technology, which is applied in the field of new multiple nucleic acid sequence detection methods and kits, can solve the problems of high equipment and site requirements, and achieve the effects of reducing hardware equipment requirements, fast speed, and high sensitivity

Inactive Publication Date: 2015-08-12
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first purpose provided by the present invention is to combine isothermal amplification technology with nanotechnology, establish a new type of visual detection method for multiple nucleic acid molecules, and solve the defects of existing nucleic acid detection technology that require high equipment and venues;

Method used

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  • Multiplex nucleic acid visualization detection method and kit based on solid phase rolling circle amplification and particle aggregation
  • Multiplex nucleic acid visualization detection method and kit based on solid phase rolling circle amplification and particle aggregation
  • Multiplex nucleic acid visualization detection method and kit based on solid phase rolling circle amplification and particle aggregation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Detection of Foodborne Pathogens

[0071] 1. Samples and reagents

[0072] Genomic DNA of Salmonella, Escherichia coli O157:H7, Vibrio cholerae and Campylobacter jejuni were purchased from ATCC;

[0073] phi29 polymerase and Taq DNA ligase were purchased from NEB (New England Biolabs);

[0074] Other reagents were of domestic analytical grade.

[0075] 2. Primers and probes

[0076] Salmonella invA gene padlock probe sequence such as 5'-SEQ ID NO.1;

[0077] Salmonella amplification primer sequence such as 5'-NH 2 (Cl 2 )-SEQ ID NO.2;

[0078] Escherichia coli O157: H7rfbE gene padlock probe sequence such as 5'-SEQ ID NO.3;

[0079] Escherichia coli amplification primer sequence such as 5'-NH 2 (Cl 2 )-SEQ ID NO.4;

[0080] Vibrio cholerae OMPW gene padlock probe sequence such as 5'-SEQ ID NO.5;

[0081] Vibrio cholerae lock amplification primer sequence such as 5'-NH 2 (Cl 2 )-SEQ ID NO.6;

[0082] Campylobacter jejuni ceuE gene padlock probe s...

Embodiment 2

[0093] Example 2: SNP typing detection of rs 1801133 and rs 1801131 loci in the human genome

[0094] 1. Samples and reagents

[0095] Human genomic DNA was extracted from peripheral blood samples by conventional phenol-chloroform method;

[0096] phi29 polymerase and Taq DNA ligase were purchased from NEB (New England Biolabs);

[0097] Other reagents were of domestic analytical grade.

[0098] 2. Primers and probes

[0099] rs1801133 probe sequence:

[0100] Wild type such as 5'-SEQ ID NO.9;

[0101] Mutant type such as 5'-SEQ ID NO.10;

[0102] Wild-type amplification primer sequence such as 5'-NH 2 (Cl 2 )-SEQ ID NO.11;

[0103] Mutant amplification primer sequences such as 5'-NH 2 (Cl 2 )-SEQ ID NO.12;

[0104] A1298C padlock probe sequence:

[0105] Wild type such as 5'-SEQ ID NO.13;

[0106] Mutant type such as 5'-SEQ ID NO.14;

[0107] Wild-type amplification primer sequence such as 5'-NH 2 (Cl 2 )-SEQ ID NO.15;

[0108] Mutant amplification primer seq...

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Abstract

The invention discloses a multiplex nucleic acid visualization detection method and kit based on solid phase rolling circle amplification and particle aggregation. The provided method utilizes the technologies of solid phase rolling circle amplification on a sheet and magnetic particle aggregation to achieve the visualization detection on multiplex nucleic acid characteristic sequences of a sample. The provided method has the characteristics of high detection flux, high sensitivity, quick detection speed, and simple operation. Furthermore, the method does not need expensive thermal amplification cycler or fluorescence detection system, can be used in medical service platforms in different levels, and are especially suitable for different medical departments in remote areas or developing areas.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a novel multiple nucleic acid sequence detection method and kit. Background technique [0002] With the DNA double helix structure model proposed by Watson and Crick and the completion of the Human Genome Project, scientists are gradually opening the door to clinical application of genome information carried by individuals, and personalized medicine has emerged as the times require. Individualized diagnosis and treatment can comprehensively grasp the individualized information of patients from the molecular level, and use this information to "tailor-made" disease prevention, diagnosis and treatment. The implementation of individualized medicine will greatly improve the health level of human beings, thus becoming the direction of medical development in the new century. To realize individualized medicine, it is first necessary to realize "individualized diagnosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6827C12Q2531/125C12Q2537/143C12Q2600/16
Inventor 李松刘洪娜何农跃邓燕张元颍
Owner SOUTHEAST UNIV
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