Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Diagnostic kit for Streptococcus iniae molecule and detection method

A technique for Streptococcus iniae and molecular diagnosis, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of inability to amplify positive fragments of standard strains, and achieve the effect of improving sensitivity

Inactive Publication Date: 2009-11-25
SUN YAT SEN UNIV
View PDF0 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, we also found that a pair of primers designed by Berridge et al. in 1998 were all negative when used to identify the Chinese strains we collected, that is, there was no amplified band
Moreover, the standard strain described in its article cannot be amplified into positive fragments

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Diagnostic kit for Streptococcus iniae molecule and detection method
  • Diagnostic kit for Streptococcus iniae molecule and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Molecular diagnostic kit for fish pathogen Streptococcus iniae

[0034] The kit consists of the following parts (10 samples)

[0035] (1) Infected tissue lysis buffer A, 1 tube, 2ml / tube, containing infected tissue total DNA extraction lysis buffer: 20mM Tris-HCl, 5mM EDTA Na 2 , 400mM NaCl, 1% SDS, pH 8.0;

[0036] (2) DNA extraction solution B, 1 tube, 20μl / tube, containing proteinase K (10mg / ml)

[0037] (3) DNA extraction solution C, 1 tube, 2ml / tube, filled with phenol-chloroform-isoamyl alcohol (25:24:1);

[0038] (4) DNA extraction solution D, 1 tube, 2ml / tube, filled with isopropanol;

[0039] (5) Positive template control solution E, 1 tube, 10 μl / tube, containing Streptococcus iniae genomic DNA;

[0040] (6) PCR reaction solution F solution, 1 tube, 250μl / tube, containing PCR amplification reaction solution, including ddH 2 O, containing Mg 2+ 10×Buffer, dNTP, primer P1, primer P2 and TaqE;

[0041] (7) box;

[0042] The DNA sequences of a p...

Embodiment 2

[0052] Embodiment 2: Specific detection of fish pathogenic bacteria Streptococcus iniae molecular diagnostic kit Use the kit in Example 1, follow the steps below:

[0053] (1) Take Streptococcus iniae, Streptococcus agalactiae, Streptococcus dysgalactiae, Pasteurella pisces, Nocardia spp., Lactococcus, Staphylococcus aureus, Vibrio anguillarum, Vibrio alginolyticus, Hydrophilic Add 200 μl lysis Buffer A to single colonies of Aeromonas and Vibrio cholerae and mix well;

[0054] (2) Add 2 μl of DNA extraction solution B, and lyse in a water bath at 50-55°C for 1 hour;

[0055] (3) Add 200 μl DNA extraction solution C to the lysate, mix well, centrifuge for 5 minutes (10000g) and take the supernatant;

[0056] (4) Add 200 μl of DNA extraction solution D to the supernatant, ice bath for 15 minutes, centrifuge for 5 minutes (10000 g), wash the precipitate twice with 70% ethanol, and dissolve the precipitate in 20 μl of sterile water as a PCR template;

[0057] (5) Take the templa...

Embodiment 3

[0065] Example 3: Detection of Streptococcus iniae Molecular Diagnostic Kit on Fish Tissue Infected with Streptococcus iniae and Fish Not Infected with Streptococcus iniae

[0066] Using the kit in Example 1, proceed as follows:

[0067] (1) Take about 0.1-0.2 g of the brain, liver, spleen, kidney or muscle tissue samples of fish infected with Streptococcus iniae and fish uninfected with Streptococcus iniae, add 200 μl of lysis BufferA, and homogenize in a homogenizer pulp;

[0068] (2) Add 2 μl of DNA extraction solution B, and lyse in a water bath at 50-55°C for 1 hour;

[0069] (3) Add 200 μl DNA extraction solution C to the lysed tissue fluid, mix well, centrifuge for 5 minutes (10000g) and take the supernatant;

[0070] (4) Add 200 μl of DNA extraction solution D to the supernatant, ice bath for 15 minutes, centrifuge for 5 minutes (10000 g), wash the precipitate twice with 70% ethanol, and dissolve the precipitate in 20 μl of sterile water as a PCR template;

[0071] ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a diagnostic kit for a Streptococcus iniae molecule and a detection method. The kit comprises lysis buffer solution A, DNA extraction B solution, DNA extraction C solution, DNA extraction D solution, positive template contrast E solution and PCR reaction solution F solution. The kit and the detection method consist of a pair of specific primers designed by a conserved sequence in a Streptococcus iniae gene group as a main body. Meanwhile, the invention optimizes the DNA extraction lysis solution and PCR reaction conditions, including magnesium ion concentration, annealing temperature and recurrent number. The kit can carry out qualitative detection on a specific DNA fragment of the Streptococcus iniae, is simple, convenient and rapid, has good specificity and high sensitivity, can be used for bacterium tracking detection of each cultivating process for fish cultivation, and has higher practical value.

Description

technical field [0001] The invention relates to a detection technology of a pathogen of aquatic animals, in particular to a molecular diagnosis kit and a detection method of a cultured fish pathogen Streptococcus iniae. Background technique [0002] Streptococcus iniae disease is an important bacterial disease of fish, which often causes explosive fish diseases in cultured fish such as tilapia and perch, leading to mass death of fish schools. Furthermore, Streptococcus iniae can also infect humans through wounds, and has been established as a new zoonotic pathogen. Nevertheless, there are many problems in the identification of Streptococcus iniae. First of all, Streptococcus iniae was discovered late, and it was only established in 1976. Therefore, many commercial biochemical identification systems did not include the bacteria, such as the more famous biochemical identification systems BioMerieux Vitek, Microscan, Vitek and ATB Expression system and so on. In Roach's 2006...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14
Inventor 李安兴周素明樊媛
Owner SUN YAT SEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com