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Specific-nucleic-acid-probe dot-blot-hybridization detection kit for tilapia streptococcus agalactiae

A detection kit, a technology for Streptococcus nias, which is used in the determination/inspection of microorganisms, microorganisms, biochemical equipment and methods, etc., can solve the problems of low positive rate, easy to be affected by error factors, and time-consuming.

Inactive Publication Date: 2015-05-06
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies of the existing Streptococcus agalactiae detection technology, the purpose of the present invention is to provide a probe dot hybridization detection kit for Streptococcus agalactiae in tilapia, aiming to solve the problem of the long time-consuming and low positive rate of the existing detection methods. , high conditions, susceptible to the problem of error factors

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: The kit of the present invention detects the sensitivity of Streptococcus agalactiae

[0055] (1) Sample: Streptococcus agalactiae from tilapia.

[0056] (2) Sample treatment: extract Streptococcus agalactiae DNA (original concentration: 364.5 μg / mL) according to the conventional method of molecular cloning, or directly boil the strain at 100°C, bathe in ice for 3 minutes, centrifuge to take the supernatant, and make it double than dilution.

[0057] (3) Take 2 μL of nucleic acid sample and spot it on a nylon membrane of appropriate size (the nylon membrane is marked with a grid, and the length and width are marked).

[0058] (4) Simply wash the membrane in 2ⅹSSC, and bake the nylon membrane at 120° for 30min. (can be stored at 4°C for later use)

[0059] (5) Put the transferred nylon membrane (spotting side up) into an appropriate volume of DIG Easy Hyb buffer (preheated to 40°C), shake gently for 30 minutes, and perform pre-hybridization.

[0060] (6...

Embodiment 2

[0074] Embodiment 2: The kit of the present invention detects the specificity of Streptococcus agalactiae

[0075] (1) Samples: 5 strains of Streptococcus agalactiae samples from tilapia and 12 strains of other bacterial samples (including Streptococcus iniae, Aeromonas hydrophila, Aeromonas temperatus, Edwardsiella tarda, Coccus aureus, Staphylococcus aureus, Proteus vulgaris, Lactobacillus, Aeromonas punctatus, etc.).

[0076] (2) Take 1 mL of bacterial liquid, centrifuge, dissolve in 100 μL of sterile water, bathe in water at 100°C for 15 minutes, immediately bathe in ice for 3 minutes, centrifuge slightly, and take the supernatant for later use.

[0077] (3) Take 2 μL of nucleic acid sample and spot it on a nylon membrane of appropriate size (the nylon membrane is marked with a grid, and the length and width are marked).

[0078] (4) Simply wash the membrane in 2ⅹSSC, and bake the nylon membrane at 120°C for 30min. (can be stored at 4°C)

[0079] (5) Put the transferred...

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Abstract

The invention relates to a specific-nucleic-acid-probe dot-blot-hybridization detection kit for tilapia streptococcus agalactiae and belongs to aquatic product biologicals. According to the kit disclosed by the invention, digoxigenin-labeled streptococcus agalactiae probes are synthesized from specific primers through PCR, and through dot molecular hybridization, the probes can be used for detecting whether streptococcus agalactiae specific nucleic acid exists in a pathological specimen or a supernatant of boiled strains or not and are used for judging whether streptococcus agalactiae infection exists or not. According to the kit disclosed by the invention, a pathological specimen tissue DNA extract or a supernatant of water-boiled strains to be detected is subjected to dot molecular hybridization, and detection and result reporting can be completed within 16-24 hours, so that the specificity is strong, and the accuracy is high.

Description

technical field [0001] The invention relates to a tilapia Streptococcus agalactiae-specific nucleic acid probe dot hybridization detection kit, which belongs to the field of aquatic biological products. Background technique [0002] Streptococcus agalactiae (Streptococcus agalactiae), also known as group B streptococci (GBS), is a class of Gram-positive bacteria widely present in nature, with a wide range of hosts, and can be effective on mammals, reptiles, amphibians and fish cause harm. In 1958, streptococcosis was first discovered in Japanese cultured rainbow trout (Oncorhychus mikiss), and the reports of fish streptococcosis gradually increased thereafter. Since 2009, the disease has been prevalent in farmed tilapia in my country. It has strong infectivity, high mortality, and difficult treatment. How to quickly, accurately and sensitively detect Streptococcus agalactiae in the early stage of infection has become a research hotspot. [0003] The current laboratory dete...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12R1/46
CPCC12Q1/6816C12Q2563/131C12Q2563/125
Inventor 可小丽卢迈新刘志刚朱华平高风英曹建萌王淼
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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