Culture-independent semiquantitative detection kit of tilapia streptococcus agalactiae

A semi-quantitative detection technology for Streptococcus lactis, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problem of low detection sensitivity, the inability to detect the content of Streptococcus agalactiae, and the inability to do culture-free detection and other issues to achieve the effect of improving the detection sensitivity

Active Publication Date: 2014-04-02
GUANGXI UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current published patents and papers, there are reports on the detection of Streptococcus agalactiae using molecular diagnostic methods. Although these methods can specifically detect Streptococcus agalactiae, they have obvious disadvantages: (1) The detection sensitivity is low, and most The situation is only applicable to the detection of bacteria after pure culture, and no culture-free detection can be achieved; (2) Some detection methods with high sensitivity, such as the LAMP method, can only qualitatively determine whether tilapia is infected with Streptococcus agalactiae, while for The degree of infection, i.e. the level of Streptococcus agalactiae in the tilapia cannot be detected

Method used

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  • Culture-independent semiquantitative detection kit of tilapia streptococcus agalactiae
  • Culture-independent semiquantitative detection kit of tilapia streptococcus agalactiae
  • Culture-independent semiquantitative detection kit of tilapia streptococcus agalactiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] 1. Draw a standard reaction comparison table:

[0066] 1.1. The first round of PCR reaction:

[0067] Take a certain amount of reagent D and mix reagent E (reagent E is a plasmid containing cfb gene, and the copy number of cfb gene is 1×108 copies / ml), and prepare cfb Gene copy numbers were 2 × 10 7 copy / ml, 2×10 6 copy / ml, 2×10 5 copy / ml, 2×10 4 copy / ml, 2×10 3 copy / ml, 2×10 2 copy / ml, 0 copy / ml cfb Gene solution.

[0068] Take fresh blood from healthy tilapia and immediately combine it with the above cfb The gene solutions were mixed in equal volumes to obtain a mixed solution.

[0069] Take 1 μl of the above mixed solution as a template, mix it with 12.5 μl of reagent A and 11.5 μl of deionized water to make a PCR reaction solution with a total volume of 25 μl, and perform the first round of PCR reaction. The reaction conditions are:

[0070] Pre-denaturation at 94°C for 5 minutes;

[0071] Denaturation at 94°C for 30s;

[0072] Anneal at 50°C for 30...

Embodiment 2

[0109] 1. Draw a standard reaction comparison table:

[0110] 1.1. The first round of PCR reaction:

[0111] Take a certain amount of reagent D and reagent E (reagent E is a plasmid containing cfb gene, and the copy number of cfb gene is 1×108 copies / ml) and mix to prepare cfb Gene copy numbers were 2 × 10 7 copy / ml, 2×10 6 copy / ml, 2×10 5 copy / ml, 2×10 4 copy / ml, 2×10 3 copy / ml, 2×10 2 copy / ml, 0 copy / ml cfb Gene solution.

[0112] Take fresh blood from healthy tilapia and immediately combine it with the above cfb The gene solutions were mixed in equal volumes to obtain a mixed solution.

[0113] Take 1 μl of the above mixed solution as a template, mix it with 12.5 μl of reagent A and 11.5 μl of deionized water to make a PCR reaction solution with a total volume of 25 μl, and perform the first round of PCR reaction. The reaction conditions are:

[0114] Pre-denaturation at 94°C for 5 minutes;

[0115] Denaturation at 94°C for 30s;

[0116]Anneal at 50°C for 3...

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Abstract

The invention relates to a culture-independent semiquantitative detection kit of tilapia streptococcus agalactiae, which consists of a reagent A, a reagent B, a reagent C, a reagent D, a reagent E, a product specification and a test result record card. The kit can be used for directly detecting whether blood of tilapia carries the streptococcus agalactiae without pure culture by a method provided by the invention, and semiquantitatively detect the quantity of the streptococcus agalactiae in the blood.

Description

technical field [0001] The invention relates to a culture-free semi-quantitative detection kit for tilapia streptococcus agalactiae. Background technique [0002] Tilapia is one of the main economic fishes in the world. It has a series of advantages such as fast growth, strong disease resistance and stress resistance, good meat quality, strong fecundity, wide adaptability and high population yield. It has become an export-oriented fish in southern provinces. The fish with the largest farming scale accounts for 50% of the world's output. The tilapia industry is of great significance for increasing employment opportunities and increasing farmers' income. However, in recent years, due to high-density breeding, population degradation, and the deterioration of water quality, environment and climate, various diseases often break out in large areas, seriously affecting the healthy development of the tilapia industry. From 2009 to 2013, a very serious tilapia epidemic broke out in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12Q1/06
CPCC12Q1/6848C12Q2549/119C12Q2545/113
Inventor 黎娅罗福广黄杰易弋伍时华
Owner GUANGXI UNIVERSITY OF TECHNOLOGY
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