Escherichia coli nucleic acid assay method using RNA constant temperature amplification

A constant temperature amplification technology for Escherichia coli, applied in the field of environmental specimen detection, can solve the problems of time-consuming, labor-intensive, detection impact, complicated operation process, etc.

Inactive Publication Date: 2020-10-13
THE FIRST INST OF OCEANOGRAPHY SOA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Due to the operating characteristics of the ship, the traditional microbial detection usually uses the agar plate culture method. The operation process is complicated and time-consuming. It usually takes 2-3 days to complete, which cannot meet the requirements of rapid detection.
DuPont, Mérieux, Merck and other international multinational companies have invested heavily in the rapid microbial detection market, and have developed such 3D, and and other rapid detection systems, the product application environment is mostly food detection, and the detection of complex seawater samples is affected to a certain extent

Method used

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  • Escherichia coli nucleic acid assay method using RNA constant temperature amplification
  • Escherichia coli nucleic acid assay method using RNA constant temperature amplification

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Effect test

Embodiment 1

[0016] Embodiment 1: the development of Escherichia coli (Ec) nucleic acid detection kit (RNA constant temperature amplification) detection reagent

[0017] 1. Design of capture probes, primers and target nucleic acid detection probes: by comparing and analyzing the existing Escherichia coli nucleic acid sequences in the Genbank database and the nucleic acid sequences reported in published literature at home and abroad, the 16SrRNA of Escherichia coli Conserved gene fragments are the target sites for amplification, and primers and probes are designed for highly conserved segments with no secondary structure.

[0018] 2. Design of internal standard detection probe: According to the principle that the competitive internal standard probe should have the same GC content and similar Tm as the detection probe, design an internal standard probe that is completely different from the target detection probe sequence and has a different fluorescent label. Needle.

[0019] 3. Constructio...

Embodiment 2

[0035] Embodiment 2: Escherichia coli (Ec) nucleic acid detection kit (RNA constant temperature amplification) and its use

[0036] 1. Take 1ml of seawater sample into a sterile sample tube, add an equal volume of sample preservation solution, vortex and oscillate for 1min, make it fully mixed, and store for later use.

[0037] 2. Prepare the sample processing tube (1.5ml centrifuge tube, the quantity is: the number of samples to be tested + 5), and mark the number of the sample to be processed and the positive control and negative control respectively;

[0038] 3. Divide the sample processing tubes in step 2 above into 3 groups, which are sample group, positive standard group and negative control group. Add 200ul of nucleic acid extraction solution to each treatment tube; add 250ul of positive standards of different concentrations to the positive standard group; continue to add 250ul of seawater samples to the sample group, and continue to add 250ul of negative standard to th...

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Abstract

The technical problem to be solved by the present invention is to provide an escherichia coli detection kit using RNA constant temperature amplification. The detection kit has an analytical sensitivity of 0.25 CFU/reaction for detecting escherichia coli. The detection kit has high specificity and high sensitivity. Amplification product RNA is easy to degrade in natural environment and has little pollution.

Description

technical field [0001] The invention relates to the technical field of environmental specimen detection, in particular to a kit for detecting Escherichia coli by using magnetic bead-RNA enrichment technology to extract and purify target RNA and constant temperature nucleic acid simultaneous amplification detection technology (SAT). The test kit of the invention can realize the detection of Escherichia coli in samples such as ship's ballast water, and quickly and accurately evaluate the safety of its discharge. Background technique [0002] The ship's ballast water is loaded in the cabin for the stability of the ship's navigation process. Ballast water contains thousands of aquatic or marine micro-organisms, plants and animals from different parts of the world, loaded in one place and discharged to another by a ship's global shipping, transporting it around the globe. Discharge of untreated ballast water at a ship's destination could introduce new invasive marine species. H...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12R1/19
CPCC12Q1/6844C12Q1/689C12Q2600/166C12Q2521/107C12Q2521/119C12Q2565/519C12Q2545/101C12Q2523/308
Inventor 高伟徐希媛郑立居金良崔振玲张帝
Owner THE FIRST INST OF OCEANOGRAPHY SOA
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