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Vibrio vulnificus isothermal amplification primer, probe, kit and detection method

A technology for isothermal amplification and Vibrio vulnificus, applied in the fields of warm amplification primers, kits and detection, and probes, to achieve the effects of improving detection sensitivity, ensuring accuracy, and good specificity

Pending Publication Date: 2021-04-13
GUANGZHOU SAGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the kit provided by the present invention can specifically enrich the target sequence by targeting the capture probe, which can improve the specificity; at the same time, the capture probe contains the T7 promoter sequence, and the sequence to be tested can be amplified through transcription and reverse transcription reactions, It can greatly improve the detection sensitivity, and the operation is simple and quick. It can solve the problem of inhibition of the isothermal amplification of the recombinase by the complex sample background, solve the practical application problem of the isothermal amplification of the recombinase, and has a wide range of application scenarios.

Method used

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  • Vibrio vulnificus isothermal amplification primer, probe, kit and detection method
  • Vibrio vulnificus isothermal amplification primer, probe, kit and detection method
  • Vibrio vulnificus isothermal amplification primer, probe, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of Capture Magnetic Beads

[0029] The preparation system of capture magnetic beads is as follows:

[0030]

[0031] The preparation method of capturing magnetic beads comprises the following steps:

[0032] (1) After preparing the above preparation system, vortex and mix, incubate at 37°C for 4 hours, and vortex and mix once every 30 minutes;

[0033] (2) After the reaction, centrifuge to collect the magnetic beads on the cap and wall of the tube, place it on a magnetic stand, and discard the supernatant;

[0034] (3) Add 500μL 1mol / L NaHCO 3 Wash twice at room temperature;

[0035] (4) Add 500μL 1mol / L NaHCO 3 Incubate for 30 minutes;

[0036] (5) Add 500 μL of nuclease-free water to wash once at room temperature, and discard the waste liquid;

[0037] (6) Add 500 μL of 0.01% Proclin to resuspend the magnetic beads to obtain capture magnetic beads and store them at 4°C.

Embodiment 2

[0038] Example 2 Sample Targeted Enrichment

[0039] Perform sample pretreatment, extract sample DNA, add capture magnetic beads prepared in Example 1, vortex and mix, incubate at 92°C for 10 minutes, centrifuge, absorb on magnetic stand for 3 minutes, discard waste liquid, and wash with nuclease-free deionized water , adding nuclease-free deionized water as a nucleic acid preservation solution to obtain targeted-enriched magnetic capture beads, and the mass concentration of the targeted-enriched magnetic capture beads is 1%.

Embodiment 3

[0040] Example 3 Recombinase Isothermal Amplification Detection

[0041] Amplification system: 60mmol / L Tris (pH 7.2), 6mmol / L dithiothreitol, 5% polyethylene glycol, 5mmol / LATP, 1.5mmol / L dNTPs, 100μmol / L phosphoinositide, 50ng / μL single Strand binding protein SSB, 10ng / μL RecQ helicase, 50ng / μL UvsX recombinase, 50ng / μL UvsY recombinase, 70ng / μL BSU DNA polymerase, 10U RNase H, 1U exonuclease III, 1μmol / L primer F, 1 μmol / L primer R, 0.8 μmol / L modified probe, 20 mmol / L magnesium acetate. After the preparation is complete, take 35 μL of the amplification system and add 15 μL of targeted enrichment capture beads.

[0042] Detection procedure:

[0043] step program temperature time number of cycles 1 warm up 40℃ 1s 1 2 Extension and Fluorescence Collection 40℃ 30s 40

[0044] The results are automatically saved at the end of the reaction. Use the original curve and endpoint fluorescence value to analyze and judge the results accordin...

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Abstract

The invention discloses a vibrio vulnificus isothermal amplification primer which comprises an upstream primer and a downstream primer. The sequence of the upstream primer is SEQ ID NO: 1. The sequence of the downstream primer is SEQ ID NO: 2. The invention belongs to the technical field of biological detection. The primer and a probe provided by the invention have good specificity, and a kit provided by the invention specifically enriches a target sequence through the targeted capture probe, so that the specificity can be improved. Meanwhile, the capture probe contains a T7 promoter sequence, the to-be-detected sequence is amplified through transcription and reverse transcription reactions, the detection sensitivity can be greatly improved, the operation is simple, convenient and rapid, the problem of inhibition of a complex sample background on recombinase isothermal amplification can be solved, the practical application problem of recombinase isothermal amplification is solved, and the wide application prospect is achieved.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a vibrio vulnificus isothermal amplification primer, a probe, a kit and a detection method. Background technique [0002] Vibrio vulnificus (vibrio vulnificus) is a bacterium that commonly lives in the ocean. Once infected, the onset is acute and the disease develops rapidly. Contact with seafood or seawater contaminated by Vibrio vulnificus, or being stabbed by marine fish and shellfish, etc., may cause Vibrio vulnificus to enter the human body and cause infection. Symptoms after infection with Vibrio vulnificus include vomiting, fever, diarrhea, hypotension, swelling and pain, etc. Clinically, it is easy to cause severe sepsis and limb necrosis, and antibiotic treatment is required as soon as possible. [0003] Isothermal amplification technology, also known as constant temperature amplification technology, can amplify specific DNA or RNA fragments at ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/63
CPCC12Q1/689C12Q1/6844C12Q2521/507
Inventor 周叙全冯凯霞陈瑞陈杰
Owner GUANGZHOU SAGENE BIOTECH
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