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Primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and detection method and application thereof

A technology of delayed Edwards, primer sequences, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of inability to accurately distinguish pathogenic and non-pathogenic, indistinguishable, inaccurate identification of Edwardsiella Bacteria and other problems, to achieve the effect of great application value, improve efficiency, and save reagents

Active Publication Date: 2011-08-03
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the results of comparative analysis of the whole genome sequence, the genome sequences of Edwardsiella bacteria have a high degree of similarity. The PCR-based method currently reported for the detection of Edwardsiella has the following problems: (1) It cannot be obtained from other microorganisms at the species level. (2) cannot accurately distinguish between Edwardsiella tarda and Edwardsiella catfish in bacteria of the genus Edwardsi; (3) cannot accurately distinguish between pathogenic and non-pathogenic Edwardsiella Bacteria

Method used

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  • Primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and detection method and application thereof
  • Primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and detection method and application thereof
  • Primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and detection method and application thereof

Examples

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Effect test

Embodiment 1

[0028] Rapid differential detection of Edwardsiella tarda:

[0029] This embodiment utilizes the multiplex PCR method to pass a PCR reaction in a PCR reaction tube to quickly identify and detect Edwardsiella tarda from diagnostic samples containing Edwardsiella tarda, that is, to quickly identify and detect Edwardsiella tarda from each bacterial strain provided in Table 1. Differentiation and detection of Edwardsiella tarda, wherein the various strains can refer to the reports in the relevant literature to obtain from the market circulation channels, for this purpose to provide optimal primer pair SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 And SEQ ID No.4, the preferred PCR reaction system provided is: 1×PCR buffer, 200μM / L dNTP, 50mM / L MgCl 2 , 1.0U Taq enzyme, 0.4μM / L SEQ ID No.1, 0.4μM / L SEQ ID No.2, 0.2μM / L SEQ ID No.3, 0.2μM / L SEQ ID No.4, 1.0μl template, Add distilled water to 25 μl. The preferred PCR reaction conditions are as follows: pre-denaturation at 94°C for 3-5 m...

Embodiment 2

[0036] Pathogenic bacterial strains were identified from Edwardsiella tarda strains, that is, positive Edwardsiella tarda screened from Example 1 was used as a template.

[0037] This embodiment utilizes the single-plex PCR method to identify pathogenic bacterial strains from Edwardsiella tarda bacterial strains, and the preferred sequence primers provided for this purpose are paired to SEQ ID No.5 and SEQ ID No.6, and the preferred PCR reaction system provided For: 1×PCR buffer, 200μM / L dNTP, 50mM / L MgCl 2 , 1.0U Taq enzyme, 0.4 μM / L SEQ ID No.5, 0.4 μM / L SEQ ID No.6, 1.0 μl template, add distilled water to 25 μl. The preferred PCR reaction conditions are as follows: pre-denaturation at 94°C for 3-5 minutes; denaturation at 94°C for 45 seconds; annealing at 50-62°C for 45 seconds; extension at 72°C for 50 seconds-1 minute and 30 seconds; Extend for 5-10 minutes. Provide a preferred qualitative observation method: carry out 1-1.5% agarose gel electrophoresis on the amplified...

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Abstract

The invention relates to the field of biotechnology, in particular to primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and a detection method and application thereof. In the method for polymerase chain reaction (PCR) detection through primers, primers for detecting pathogenic and nonpathogenic Edwardsiella and a method for single or multiplex amplification detection by applying the primers are further related; and the invention further relates to a kit comprising the primers and used for detecting the Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda. By adopting multiplex PCR amplification, more than two pairs of primers are present in the same reaction system and can amplify more than two target genes, so that the multiplex PCR is reagent-saving, time-saving and labor-saving than single PCR; therefore, the cost is reduced and the efficiency is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer sequence for detecting Edwardsiella tarda, Edwardsi catfish or pathogenic Edwardsiella tarda, a detection method and application thereof. Background technique [0002] Edwardsiella tarda (Edwardsiella tarda) is a common pathogenic bacterium in aquatic animals. It infects a wide range of hosts, including amphibians (toads, frogs), reptiles (lizards, snakes, turtles, crocodiles), fish (eels, catfish, etc.) , flounder, etc.), birds (penguins, vultures, ostriches) and mammals including humans (sea lions, manatees, pigs, dogs, cows, monkeys). Edwardsiella tarda can survive in seawater and freshwater environments, infect a variety of wild and farmed fish, and cause Edwardsiellosis with hemorrhagic sepsis as the main symptom. With the rapid development of aquaculture in my country, Edwardsiella tarda not only affects the cultivation of a variety of freshwater economic animals (eel...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 莫照兰李贵阳肖鹏李杰
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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