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General detection method for pathogenic vibrios and nucleic acid isothermal amplification detection kit used in same

A technology of Vibrio nucleic acid and isothermal amplification, applied in the field of marine aquaculture animal pathogen detection, can solve problems such as difficulty in meeting the control of marine aquaculture animal diseases, difficulty in on-site batch detection, false positives in fresh samples, etc. Effectiveness of detection, avoidance of false positive results, avoidance of contamination

Inactive Publication Date: 2011-08-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current vibrio detection method is aimed at a single sample, and the operation steps are cumbersome, it is difficult to achieve on-site batch detection, and it is difficult to meet the needs of marine animal disease prevention and control
Currently, there are three main methods for the detection of pathogenic Vibrio: ①Physiological and biochemical identification method, which is cumbersome and time-consuming; detection and other characteristics, but false positives are prone to occur when testing fresh samples, which limits its application in production practice to a certain extent; ③ polymerase chain reaction (PCR), which is fast and sensitive, but requires expensive Nucleic acid amplification instrument, which also limits the application in production practice

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1. A general detection kit for the isothermal amplification of pathogenic Vibrio nucleic acid in seawater cultured animals (5 samples, which can be used for the detection of 5 samples), consisting of the following contents:

[0050] (1) Grinding solution tube, 1 tube, 3 ml.

[0051] Grinding fluid preparation: 10 parts of 1 M Tris-HCl solution (Tris-HCl, pH 8.0), 0.25 M ethylenediaminetetraacetic acid disodium solution (EDTANa 2 ) 2 parts, mass fraction is 10 parts of sodium dodecylsulfonate solution (SDS) of 5%, 0.5 part of mercaptoethanol, 0.01 part of 8-hydroxyquinoline, 7 parts of balanced phenols, 8 parts of chloroform, with double distilled water Make up to 100 servings. The above parts are all parts by volume.

[0052] (2) Tube A of nucleic acid extraction solution, 1 tube, containing 400 μl of 3M sodium acetate solution (pH 5.2).

[0053] (3) Nucleic acid extraction solution tube B, tube l, filled with 10 ml of absolute ethanol.

[0054] (4) Tube ...

Embodiment 2

[0077] Embodiment 2. A universal detection kit (5 samples) for the isothermal amplification of pathogenic Vibrio nucleic acid in mariculture animals. Only the following content is different from embodiment 1, and the rest are the same as embodiment 1.

[0078] Grinding solution preparation: 5 parts of 2 M Tris-HCl solution (Tris-HCl, pH 8.0), 0.5 M ethylenediaminetetraacetic acid disodium solution (EDTANa 2 ) 1 part, 7 parts of sodium dodecylsulfonate solution (SDS) with a mass fraction of 7%, 0.01 part of mercaptoethanol, 0.02 part of 8-hydroxyquinoline, 5 parts of balanced phenol, 10 parts of chloroform, fixed with double distilled water Can hold up to 100 copies. The above parts are all parts by volume.

[0079] The composition of the LAMP reaction solution is as follows: LAMP primers VIBRIO-FIP and VIBRIO-BIP each 4 μM, LAMP primers VIBRIO-F3 and VIBRIO-B3 each 0.5 μM, dATP, dGTP and dCTP each 2 mM, dTTP and dUTP each 1.0 mM, Tris -HCl 40mM, KCl 20mM, (NH 4 ) 2 SO 4 ...

Embodiment 3

[0080] Example 3. A general detection kit for isothermal nucleic acid amplification of pathogenic Vibrio in mariculture animals (5 samples). Only the following content is different from Example 1, and the rest are the same as Example 1.

[0081] Grinding solution preparation: 7 parts of 1.5 M Tris-HCl solution (Tris-HCl, pH 8.0), 1.0 M ethylenediaminetetraacetic acid disodium solution (EDTANa 2 ) 0.5 part, 5 parts of sodium dodecylsulfonate solution (SDS) with a mass fraction of 10%, 0.01 part of mercaptoethanol, 0.015 part of 8-hydroxyquinoline, 10 parts of balanced phenol, 10 parts of chloroform, fixed with double distilled water Can hold up to 100 copies. The above parts are all parts by volume.

[0082] The composition of the LAMP reaction solution is as follows: 1 μM each of LAMP primers VIBRIO-FIP and VIBRIO-BIP, 0.25 μM each of LAMP primers VIBRIO-F3 and VIBRIO-B3, 0.5 mM each of dATP, dGTP and dCTP, 0.5 mM each of dTTP and dUTP, Tris-HCl 40mM, KCl 10mM, (NH 4 ) 2 S...

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PUM

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Abstract

The invention discloses a general nucleic acid isothermal amplification detection kit for pathogenic vibrios of mariculture animals. The detection kit comprises a grinding liquid tube into which grinding liquid is filled, a nucleic acid extracting solution tube A into which solution of sodium acetate is filled, a nucleic acid extracting solution tube B into which absolute ethanol is filled, a nucleic acid extracting solution tube C into which 70 mass percent ethanol solution is filled, a tris-hydrogen chloride ethylene diamine tetraacetic acid (TE) buffer solution tube into which TE buffer solution is filled, a uracil-DNA-glycosylase (UNG) tube into which uracil-DNA-glycosylase is filled, a loop-mediated isothermal amplification (LAMP) reaction liquid tube into which LAMP reaction liquid is filled, a bacillus stearothermophilus deoxyribonucleic acid (Bst DNA) polymerase tube into which Bst DNA polymerase is filled, a color-developing agent tube into which a nucleic acid dye SYBR Green I is filled, a positive control nucleic acid tube into which positive DNA of the vibrios is filled and a negative control tube into which sterilized double distilled water is filled. The invention also discloses a method for detecting the pathogenic vibrios of the mariculture animals by utilizing the detection kit.

Description

technical field [0001] The invention relates to a pathogen detection technology for seawater cultured animals, in particular to a general detection kit and a detection method for nucleic acid isothermal amplification of pathogenic Vibrio in seawater cultured animals. Background technique [0002] Pathogenic Vibrio is one of the common pathogenic bacteria groups in the marine environment, and the vibriosis caused by it is a kind of infectious disease with a wide range of epidemics and serious harm, which can harm a variety of marine cultures such as fish, crustaceans and shellfish. Animals have caused huge economic losses to the mariculture industry. With the vigorous development of mariculture industry, vibriosis occurs frequently, and the common pathogenic species has Vibrio harveyi ( Vibrio Harvey ), Vibrio alginolyticus ( V. alginolyticus ), Vibrio parahaemolyticus ( V. parahaemolyticus ), Vibrio vulnificus ( V. vulnificus ), Vibrio flexneri ( V. furnissii ), Vib...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 徐海圣何琳
Owner ZHEJIANG UNIV
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