Primers for quickly detecting Edwardsiella tarda by using gene amplification method

A technology for Edwardsiella tarda and gene amplification, which is applied in the field of primers for rapid detection of Edwardsiella tarda by gene amplification method, and can solve the problems that cannot be popularized and applied

Active Publication Date: 2012-07-11
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods have the advantages of rapidity and specificity compared with the traditional routine identification of bacteria. However, the above methods are restricted by the preparation of antigens and antibodies, and the need for special equipment such as PCR instruments and gel el

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  • Primers for quickly detecting Edwardsiella tarda by using gene amplification method
  • Primers for quickly detecting Edwardsiella tarda by using gene amplification method
  • Primers for quickly detecting Edwardsiella tarda by using gene amplification method

Examples

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Effect test

Embodiment 1

[0156] Embodiment 1: Carry out bacterial specific detection with the gene amplification primer of E.tarda

[0157] Firstly, the primers provided by the present invention are synthesized, and then the specific detection of E.tarda is carried out according to the following steps.

[0158] (1) Preparation of bacterial DNA: The DNA of bacterial samples was extracted using TIANamp Bacteria DNA Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the extracted DNA was used on DNA agarose gel Recovery Kit (Zymo, USA) was used for purification. After the DNA sample was prepared, it was placed at 95°C for 5 minutes, and then denatured in an ice bath for 5 minutes before being used as a template.

[0159] Prepare the reaction system: 0.2 μM each of primer 1 and primer 2 of the first set of primers, 1.6 μM each of primer 3 and primer 4, 0.8 μM each of primer 5 and primer 6, 1.4 mM each of dNTP, MgCl 2 6mM, Betaine 1.2M, Tris-HCl 20mM, KCl 10mM, MgSO 4 2mM, (NH 4 ) 2 SO...

Embodiment 2

[0164] Embodiment 2: Carry out bacterial specific detection with the gene amplification primer of E.tarda

[0165] First, the primers provided by the present invention are synthesized, and then the bacteria-specific detection of E. tarda is carried out according to the following steps.

[0166] (1) Preparation of bacterial DNA: The DNA of bacterial samples was extracted using TIANamp Bacteria DNA Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the extracted DNA was used on DNA agarose gel Recovery Kit (Zymo, USA) was used for purification. After the DNA sample was prepared, it was placed at 95°C for 5 minutes, and then denatured in an ice bath for 5 minutes before being used as a template.

[0167] Prepare the reaction system: 0.2 μM each of primer 1 and primer 2 of the fourth set of primers, 1.6 μM each of primer 3 and primer 4, 1.4 mM each of dNTP, MgCl 2 6mM, Betaine 1.2M, Tris-HCl 20mM, KCl 10mM, MgSO 4 2mM, (NH 4 ) 2 SO 4 10 mM, Triton X-100 0.1%...

Embodiment 3

[0172] Embodiment 3: Carry out bacterial specific detection with the gene amplification primer of E.tarda

[0173] First, the primers provided by the present invention are synthesized, and then the bacteria-specific detection of E. tarda is carried out according to the following steps.

[0174] (1) Preparation of bacterial DNA: DNA of bacterial samples was extracted using TIANamp Bacteria DNA Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. Recovery Kit (Zymo, USA) was used for purification. After the DNA sample was prepared, it was placed at 95°C for 5 minutes, and then denatured in an ice bath for 5 minutes before being used as a template.

[0175] Prepare the reaction system: 0.2 μM each of primer 1 and primer 2 of the fifth set of primers, 1.6 μM each of primer 3 and primer 4, 0.8 μM each of primer 5 and primer 6, 1.4 mM each of dNTP, MgCl 2 8mM, Betaine 1.2M, Tris-HCl 20mM, KCl 10mM, MgSO 4 2mM, (NH 4 ) 2 SO 410 mM, Triton X-100 0.1%, Bst DNA polymerase...

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Abstract

The invention discloses gene amplification primers for quickly detecting Edwardsiella trada. Specific primers are designed by taking esrB of E. tarda or a hlyB upstream sequence of the Edwardsiella trada as a target. A gene amplification detection method by the primers has the characteristics of simplicity, convenience, quickness, specificity and sensitivity; genes with 10 copy number can be detected in 40 minutes only through a water bath or a metal bath; and the method is suitable for quickly detecting the E. tarda in field and has good application prospect.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, in particular to a primer for rapid detection of Edwardsiella tarda by a gene amplification method and an application thereof. Background technique [0002] E. tarda is a Gram-negative short bacillus, Edwardsiella genus, first reported by Hoshina in 1962, and it is one of the pathogenic bacteria with great harm in the current aquaculture industry. The host range of the bacterium is very wide. In addition to infecting a variety of fish, it has also been reported to infect amphibians, reptiles and mammals (including humans). Therefore, in order to diagnose, monitor and prevent E.tarda in time, it is of great significance to establish a fast, accurate and simple E.tarda detection technology. [0003] Conventional bacterial identification methods include the identification of pathogenic morphology, physiological and biochemical levels, and protein levels. However, due to the shortcom...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/01
Inventor 黄倢谢国驷张庆利史成银王秀华杨冰刘莉
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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