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Reagent box for accurately discriminating catfish and large-mouth catfish and discrimination method

A technology for identifying catfish and largemouth catfish, which can be used in biochemical equipment and methods, microbial determination/inspection, etc., and can solve the problems of distinction and difficulty in correct identification.

Inactive Publication Date: 2007-08-29
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at the seedling stage, generally under 250g, the two catfish are very similar in shape, and it is difficult to distinguish them correctly based on their appearance characteristics, and even experienced fish taxonomy workers are also difficult to correctly identify them

Method used

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  • Reagent box for accurately discriminating catfish and large-mouth catfish and discrimination method

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Experimental program
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Effect test

Embodiment 1

[0037] The kit for identifying catfish and largemouth catfish can be used 100 times, and its composition is as follows:

[0038] (a) DNA extraction reagents, including

[0039] Reagent 1, 40ml, its composition is: 10mmol / L Tris-HCl, pH8.0; 2mmol / L EDTA, pH8.0; 0.4mol / L NaCl, 1% SDS;

[0040] Reagent 2, 0.8ml, its composition is: proteinase K, 20mg / mL;

[0041] Reagent 3, 30 ml, its composition is: sodium chloride, 6mol / L;

[0042] Reagent 4: 30ml isopropanol (analytical pure);

[0043] Reagent 5: 100ml 70% ethanol.

[0044] (b) polymerase chain reaction reagents: including reaction premix reagent 6 and reagent 7;

[0045] Reagent 6, containing 100 parts of the following components (the number in parentheses is the volume of 1 part, and the total amount of 100 parts is 2450 μl): 10 times polymerase chain reaction buffer (containing MgCl 2 15mM) (2.5μl), specific primers NS (0.5μl (10μM)) and NA (0.5μl (10μM)) (please biotechnology companies (such as Shanghai Sangong, Hands...

Embodiment 2

[0061] 1. Take 50 mg of fish fins, cut them into pieces, and add 400 μl of reagent 1;

[0062] 2. Add 10 μl of Reagent 2, and bathe in water at 60°C for about 2 hours until there are no particles in the solution;

[0063] 3. Add 300 μl of reagent 3, shake and mix for 30 seconds, then centrifuge at 10,000×g for 30 minutes, and transfer the supernatant to a new centrifuge tube;

[0064] 4. Add an equal volume of reagent 4, place at -20°C for 1 hour, then centrifuge at 10,000×g for 15 minutes, discard the supernatant;

[0065] 5. Wash the precipitate with 1ml of Reagent 5, centrifuge at 10,000×g for 5min, dry in the air, dissolve in sterilized double distilled water, and obtain the DNA extraction solution;

[0066] 6. PCR amplification: take 1 μl of DNA as the template for PCR amplification; mix 23.875 μl of reagent 6 with 0.125 of reagent 7; melt at 94°C for 3 minutes on a thermal cycler; then denature at 94°C for 30 seconds, Renature for 30 seconds, extend at 72°C for 45 seco...

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Abstract

The invention provides a reagent box of accurately identifying S.asotus and S.soldatovi and the detecting method thereof. And the reagent box comprises DNA extracting reagent, including reagents 1, 2, 3, 4 and 5; PCR reagent, including reaction premixing reagent 6 and reagent 7; electrophoresis and color-developing reagent, including reagents 8, 9, 10 and 11. And the invention espeically provides a pair of specific primers NS and NA, which are used to make PCR amplification reaction to be able to accurately identify S.asotus and S.soldatovi, and the features: accurate, batch, low damage to identified fish and unable to cause the identified fish death.

Description

technical field [0001] The invention relates to a kit for identifying catfish (Silurus asotus) and catfish in batches accurately and in batches, and a method for using the kit to identify catfish and catfish (Silurus meridionalis). Background technique [0002] Both catfish and largemouth catfish belong to the genus Silurus in classification. Catfish is a widely distributed species in the world, while largemouth catfish is mainly distributed in the Yangtze River, Minjiang River, Xiangjiang River and other water systems in my country. Generally, the water systems where largemouth catfish is distributed are all There are catfish distribution. Adult catfish (S. asotus) are much smaller than largemouth catfish (S. meridionalis), and they can be distinguished by their size and external features. However, in the seedling stage, generally under 250g, the two catfishes are very similar in shape, and it is difficult to distinguish them correctly based on their appearance characterist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张其中
Owner SOUTHWEST UNIVERSITY