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Saprolegnia spore detection reagent and detection method

A detection reagent and a technology for water mold, applied in the field of water mold spore detection reagent and detection, can solve the problems of difficulty in accuracy, time-consuming, time-consuming and labor-intensive data processing, and achieve the effects of simple operation, convenient method and low cost

Inactive Publication Date: 2012-12-19
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problems that the traditional method for detecting Saprolegnia spores or mycelium mass in aquaculture water is tedious, time-consuming, labor-intensive, low specificity, time-consuming for detection data processing and difficult to grasp the accuracy

Method used

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  • Saprolegnia spore detection reagent and detection method
  • Saprolegnia spore detection reagent and detection method
  • Saprolegnia spore detection reagent and detection method

Examples

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Embodiment example 1

[0067] The present invention is applied to the early warning of saprolegniasis, that is, the detection method of saprolegniasis spore concentration in the aquaculture water body, and its special feature is to follow the following steps:

[0068] The sample to be tested (Saprolegnia spores in the aquaculture water body) is collected, and 1mL of the water sample to be tested is drawn with a quantitative pipette, and diluted into 5 gradients by 10 times. Take 100 μL sample from tube 1 in tube 2 and add it to 900 μL double distilled water (equivalent to 10 μL of tube 1). -1 Concentration), No. 3 tube, take 100 μL sample from No. 2 tube and add 900 μL double-distilled water (equivalent to No. 1 tube 10 -2 Concentration), No. 4 tube, take 100 μL sample from No. 3 tube and add 900 μL double distilled water (equivalent to No. 1 tube 10 -3 Concentration), No. 5 tube, take 100 μL sample from No. 4 tube and add 900 μL double distilled water (equivalent to No. 1 tube 10 -4 concentration...

Embodiment example 2

[0076] The present invention is applied to the detection of suspected saprolegniasis samples, that is, the detection method of saprolegnia mycelia, and its special feature is to follow the steps below:

[0077] The sample to be tested (water mold mycelial mass on the diseased sample) is collected, and the mycelial mass at the lesion is scraped with sterilized tweezers and put into a test tube;

[0078] 1) Extract the nucleic acid of the sample to be tested, centrifuge to collect the sample to be tested (the saprolegniasis mycelium on the diseased sample), discard the supernatant, add 500 μL of nucleic acid lysate A and mix well; then add 100 μL of nucleic acid lysate B and 300 μL of nucleic acid lysate C violent shock;

[0079] 2) To carry out the detection reaction, take 20 μL of reaction buffer and add 3 μL of the nucleic acid extraction solution in step 1;

[0080] 3) After reacting at 60°C for 45 minutes, add 2 μL of reaction chromogenic solution. If the reaction turns gr...

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Abstract

The invention relates to the fungus detection technology and provides a Saprolegnia detection reagent. The Saprolegnia detection reagent comprises the following components: (1) a detection reaction solution which contains 20mmol / L of Tri-HCl, 10mmol / L of (NH4)2SO4, 10mmol / L of KCl, 0.1%Triton X-100, 10mmol / L of MgSO4, 1mmol / L of dNTPs (deoxyribonucleoside triphosphates), 0.6mmol / L of Betaine, 1.5mu mol / L of inner primers FIP / BIP, 0.8mu mol / L of outer primers F3 and B3 and 8U of BstDNA polymerase large fragments; (2) reaction coloring liquid which contains a 10% SYBR Green fluorescent dye / TAE buffer solution; and (3) nucleic acid lysis buffer which contains a nucleic acid lysis solution A, a nucleic acid lysis solution B and a nucleic acid lysis solution C, wherein the nucleic acid lysis solution A contains 100mmol / L of Tris HCl and 40mmol / L of ethylene diamine tetraacetic acid (EDTA), the pH value of Tris HCl is 9.0 and the pH value of EDTA is 8.0; the nucleic acid lysis solution B contains 10% sodium-dodecyl sulphate (SDS); and the nucleic acid lysis solution C contains 100% benzyl chloride. The detection method is simple and is low in cost and easy in operation; and the quarantine personnel can perform in-situ sampling near the fishpond.

Description

technical field [0001] The invention relates to fungi detection technology, in particular to a saprolegniasis spore detection reagent and detection method. Background technique [0002] Saprolegniasis, also known as Dermatomyucosis, is the most common disease in freshwater fish. Saprolegniasis is the main pathogen that causes saprolegniasis in aquatic animals. It adapts to a wide temperature range and distributes in a wide range. It can cause fish disease in all seasons. Saprolegnias belong to Saprolegniales, Peronosporales and Blaslociadiales in Oomycetes, among which Saprolegnia, Cottonella ( Achlya) and Aphanomyces are the most common species and are the main pathogens in fish egg hatching, fish rearing and grow-out culture. [0003] Saprolegniasis has no strict selectivity for hosts, and occurs in all growth stages of aquatic animals. It mainly parasitizes fish wounds and dead eggs, harms farmed fish and reduces the hatching rate of fish eggs. Under appropriate condit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
Inventor 吕利群王浩张楠
Owner SHANGHAI OCEAN UNIV
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