Primer sequence and detecting reagent kit for detecting delayed Edward's bacteria

A slow Edwards, detection kit technology, applied in the field of DNA sequence, can solve the problems of laborious, time-consuming, affecting the accuracy of bacterial identification, etc., and achieve the effect of high practical value

Inactive Publication Date: 2007-04-11
天津市水产研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current identification of Edwardsiella tarda mainly relies on conventional physiological and biochemical methods, which are time-consuming, laborious, and difficult to determine the resu...

Method used

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  • Primer sequence and detecting reagent kit for detecting delayed Edward's bacteria

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Comparison scheme
Effect test

Embodiment 1

[0025] A primer sequence for detecting Edwardsiella tarda, which is composed of an upstream primer and a downstream primer. The upstream primer has the nucleotide sequence described in SEQ ID No1 in the sequence list, and the downstream primer has SEQ in the sequence list. The nucleotide sequence described in ID No2.

[0026] Sequence Listing

[0027] SEQ ID No1

[0028] 5’--GTCGGTGGATTGACGGAGGT-3

[0029] SEQ ID No2

[0030] 5’--AAGAGGGCCAGCGGGATT-3’

Embodiment 2

[0032] A test kit for Edwards sluggish bacteria, which consists of the following reagents:

[0033] Reagent A: Mix 1.25g of ordinary nutrient broth with 50mL of old sea water, and then autoclave it at 0.3Kpa;

[0034] Reagent B: Mix 2.5 μL of 10-fold concentration PCR reaction buffer, 1.25 U of Taq DNA polymerase, and 17.25 μL of sterilized double-distilled water;

[0035] Reagent C: 0.5μL of the nucleotide sequence described in 10μM SEQ ID No1, 0.5μL of the nucleotide sequence described in 10μM SEQ ID No2, 0.5μL of 10mM dNTPs, 25mM MgCl 2 1.5μL mixed and made;

[0036] Reagent D: 60ng / mL of total genomic DNA of Edwardsiella lentus;

[0037] Reagent E: Sterilized double distilled water;

[0038] Reagent F: Take 50mL 20 times TBE electrophoresis buffer, make it contain 0.09mol hydroxymethylaminomethane-boronic acid, 2mmol ethylenediaminetetraacetic acid;

[0039] Reagent G: 0.3g agarose, so that it contains 20μg ethidium bromide;

[0040] A pack of sterilized toothpicks.

[0041] P...

Embodiment 3

[0043] A test kit for Edwards sluggish bacteria, which consists of the following reagents:

[0044] Reagent A: Mix 0.5g of ordinary nutrient broth with 50mL of old sea water, and then autoclave at 0.1Kpa to make it;

[0045] Reagent B: Mix 1.5μL of 10-fold concentration PCR reaction buffer, 1U of Taq DNA polymerase, and 17μL of sterilized double-distilled water;

[0046] Reagent C: 0.2μL of the nucleotide sequence described in 10μM SEQ ID No1, 0.2μL of the nucleotide sequence described in 10μM SEQ ID No2, 0.2μL of 10mM dNTPs, 25mM MgCl 2 1.0μL mixed and made;

[0047] Reagent D: 20ng / mL of total genomic DNA of Edwardsiella lentus;

[0048] Reagent E: Sterilized double distilled water;

[0049] Reagent F: Take 50mL 20 times TBE electrophoresis buffer, make it contain 0.09mol hydroxymethylaminomethane-boronic acid, 2mmol ethylenediaminetetraacetic acid;

[0050] Reagent G: 0.3g agarose, which contains 15μg ethidium bromide;

[0051] A pack of sterilized toothpicks.

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Abstract

The present invention discloses primer sequence and detecting reagent kit for detecting delayed Edward's bacteria. The detecting reagent kit contains the following reagents: mixture of nutrient broth and aged sea water through autoclaving; mixture of PCR buffer, Taq DNA polymerase and sterile double distilled water; mixture of nucleotide sequence of SEQ ID No. 1 and SEQ ID No. 2, dNTPs and MgCl2; genome total DNA of delayed Edward's bacteria; sterile double distilled water; TBE electrophoretic buffer containing Tris-boric acid and EDTA; and agarose containing ethidium bromide. The present invention has primer sequence with optimized PCR condition and system, and the PCR detecting method for detecting delayed Edward's bacteria and the detecting reagent kit are simple, fast, sensitive and specific.

Description

Technical field [0001] The present invention relates to a DNA sequence in biotechnology, a kit for detecting a kind of bacteria using the sequence, and in particular to a primer sequence and a detection kit for detecting Edwards tarda. Background technique [0002] Edwards tarda is the only bacteria in the genus Edwards of the Enterobacteriaceae family that causes diarrhea and other diseases. It can also cause infections in many animals such as shellfish, fish, amphibians and birds. Edwards tarda is a ubiquitous bacterium in seawater, and it has been isolated from aquatic economic animals in various places. Especially in recent years, the ascites disease of seawater industrial farmed turbot and brown flounder caused by the infection of Edwards tarsus has caused huge economic losses to seawater factory farming enterprises. In addition to causing ascites disease in fish, Edwards tarsus is also an important pathogen of eel liver and kidney disease, red head disease and Edwards disea...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 孙金生薛淑霞耿绪云李翔胡金城
Owner 天津市水产研究所
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