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Rolling circle amplification-based colorimetric assay method for target nucleic acids or proteins

A technology of rolling circle amplification and target nucleic acid, applied in biochemical equipment and methods, biological testing, material inspection products, etc., can solve problems such as inability to adapt to rapid detection and complexity, and achieve intuitive and clear detection results, simple operation, and specificity good sex effect

Active Publication Date: 2012-07-18
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, some detections based on rolling circle amplification are relatively complicated and require expensive instruments, such as chip detection (whole genome rolling circle Amplified oligonucleotides, patent number: 200480027696.1) and Raman spectroscopy detection (Biomolecule analysis by rolling circle amplification and SEBS detection, patent number: 200480001922.9), obviously these methods are not suitable for rapid detection on the spot

Method used

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  • Rolling circle amplification-based colorimetric assay method for target nucleic acids or proteins
  • Rolling circle amplification-based colorimetric assay method for target nucleic acids or proteins
  • Rolling circle amplification-based colorimetric assay method for target nucleic acids or proteins

Examples

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Embodiment 1

[0039] A colorimetric detection method for target nucleic acid based on rolling circle amplification

[0040] This example describes the establishment of a method for colorimetric detection of target nucleic acids based on rolling circle amplification, taking type A H1N1 nucleic acids as an example

[0041] (1) Design and synthesis of detection probes

[0042] The present invention uses the influenza virus H1N1 (Influenza A virus H1N1envelope_2) sequence as the target gene template, and the DNA probe sequence is as follows (the synthesis of the probe is completed by Dalian TaKaRa Biotechnology Co., Ltd.).

[0043] Table 1. H1N1 DNA target sequences and probes

[0044]

[0045] (2) Labeling of capture probes on magnetic beads

[0046] Take 50 μL of unlabeled amino magnetic beads, add 50 μL of MES (0.1mol / L, pH 4.6), wash twice, magnetically separate, and discard the supernatant. Add 40 μl of MES and 10 μl of carboxy-modified capture probe, and mix thoroughly. Prepare 100...

Embodiment 2

[0055] Colorimetric detection of target protein based on rolling circle amplification

[0056] This example describes the establishment of a method for colorimetric detection of target proteins based on rolling circle amplification, taking the heart disease marker troponin as an example

[0057] 1. Design and synthesis of signal reporter primer probe

[0058]

[0059] 2 Labeling of capture antibodies on magnetic beads

[0060] Wash 50ul magnetic beads with an equal amount of MES (25Mm, PH6) twice, remove the supernatant after magnetic separation, add 25ul EDC and NHS (freshly prepared, the concentration is 50mg / ml) in turn, mix and incubate for 30 minutes, magnetic After separation, remove the supernatant and wash twice with MES; dissolve the activated magnetic beads in 45ul MES, mix well with 2.5ug antibody, and incubate for 30 minutes (slow speed to prevent magnetic beads from sedimenting); then add 50ul Tris (PH7 , 50mM) for 30 minutes to seal the unreacted groups on t...

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Abstract

The invention relates to a rolling circle amplification-based colorimetric assay method for target nucleic acids or proteins. The method mainly utilizes the hybridization between nucleic acids and antigen-antibody binding to indirectly fix the target nucleic acids or proteins to be assayed onto magnetic beads, rolling circles are added, a rolling circle primer on hybrid is utilized to carry out constant-temperature rolling circle amplification, and finally, the chromogenic reaction of nanogold is utilized to assay constant-temperature amplified macromolecular product in order to achieve the purpose of relatively assaying the target nucleic acids and proteins. By integrating rolling circle amplification and the chromogenic reaction of nanogold, the method not only amplifies a large quantity of micro-sample signals, but also realizes the rapid, visual assay of results, avoids the usage of a PCR (Polymerase Chain Reaction) instrument and other expensive instruments, and decreases the requirement on the experimental hardware condition. The method is expected to be applied in bedside rapid diagnosis and the field rapid assay of microorganisms in food safety and the environment.

Description

technical field [0001] The invention relates to a colorimetric detection method for target nucleic acid or protein based on rolling circle amplification, which belongs to the detection field of biological macromolecules. Background technique [0002] Rapid, specific, and high-throughput biomolecular detection methods are of great significance to the diagnosis of pathogenic microorganisms and genetic diseases in the fields of medical care and biosafety. The highly sensitive detection of low-concentration biomolecules directly from samples has broad and potential applications in a large number of fields such as laboratory medicine, pathology, epidemiology, environmental and food testing. At present, the most widely used amplification method is mainly polymerase chain reaction (PCR). PCR technology can realize the amplification of 10 DNA molecules, and has high sensitivity and specificity. However, its main disadvantage is that it needs to use complex The equipment and the rea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/68
Inventor 毛红菊邢亚斯王萍金庆辉赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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