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Riemerella anatipestifer detection kit and method based on loop-mediated isothermal amplification technology

A ring-mediated isothermal and Riemerella anatipestifer technology is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection. Achieve the effect of simple and fast operation, strong specificity and low detection cost

Inactive Publication Date: 2010-10-27
CHONGQING ACAD OF ANIMAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no report on the detection of Riemerella anatipestifer based on the loop-mediated isothermal amplification technique.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. The Riemerella anatipestifer detection kit based on the loop-mediated isothermal amplification technology consists of the following reagents:

[0037] a. The upstream internal primer FIP aqueous solution and the downstream internal primer BIP aqueous solution with a concentration of 5 μmol / L, the upstream external primer F3 aqueous solution and the downstream external primer B3 aqueous solution with a concentration of 20 μmol / L: the primer sequences are as follows:

[0038] Upstream inner primer FIP: 5'-caggcttccaccaccattgtccaa-tgagacacggaccagactc-3';

[0039] Downstream inner primer BIP: 5'-cgtgaaggacgacggccctat-ctaccctcacgagagtaggt-3';

[0040]Upstream outer primer F3: 5'-tttaggggggcctgagagg-3';

[0041] Downstream outer primer B3: 5'-ccggtgcttattcgtacagt-3';

[0042] b. An aqueous solution of Bst DNA polymerase with a concentration of 8 U / μl;

[0043] c, 10× thermal polymerization reaction buffer: Tris-HCl with a concentration of 250mmol / L and a pH of 8.8, pota...

Embodiment 2

[0054] 1. The Riemerella anatipestifer detection kit based on the loop-mediated isothermal amplification technology is different from the kit described in Example 1 in that the specific primer sequences are different. The primer sequences of this kit are as follows:

[0055] Upstream internal primer FIP: 5'-tattcctcactgctgcctcccg-gtgatcccccacactggt-3';

[0056] Downstream inner primer BIP: 5'-cgtgaaggacgacggccctat-ctaccctcacgagagtaggt-3';

[0057] Upstream outer primer F3: 5'-tttaggggggcctgagagg-3';

[0058] Downstream outer primer B3: 5'-ccggtgcttattcgtacagt-3'.

[0059] 2. The method of using the Riemerella anatipestifer detection kit based on the loop-mediated isothermal amplification technology to detect Riemerella anatipestifer is the same as the method described in Example 1. It was found that the color of the blank control group It is yellow, indicating that it does not contain Riemerella anatipestifer; the color of the experimental group turns green, indicating that ...

Embodiment 3

[0061] 1. The Riemerella anatipestifer detection kit based on the loop-mediated isothermal amplification technology is different from the kit described in Example 1 in that the specific primer sequences are different. The primer sequences of this kit are as follows:

[0062] Upstream internal primer FIP: 5'-caggcttccacccattgtccaa-acactggtactgagacacgg-3';

[0063] Downstream inner primer BIP: 5'-cgtgaaggacgacggccctat-ctaccctcacgagagtaggt-3';

[0064] Upstream outer primer F3: 5'-tttaggggggcctgagagg-3';

[0065] Downstream outer primer B3: 5'-ccggtgcttattcgtacagt-3'.

[0066] 2. The method of using the Riemerella anatipestifer detection kit based on the loop-mediated isothermal amplification technology to detect Riemerella anatipestifer is the same as the method described in Example 1. It was found that the color of the blank control group It is yellow, indicating that it does not contain Riemerella anatipestifer; the color of the experimental group turns green, indicating tha...

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Abstract

The invention discloses riemerella anatipestifer detection kit and method based on a loop-mediated isothermal amplification technology. Based on six specific regions of a 16S rRNA conserved region of riemerella anatipestifer, two specific primers and two specific outer primers are designed in the kit and can specifically distinguish the riemerella anatipestifer and other serotype salmonellae so as to ensure the high specificity and the reliability of a detection result of loop-mediated isothermal amplification. The invention detects the riemerella anatipestifer on the basis of the loop-mediated isothermal amplification technology, can amplify a target sequence rapidly, efficiently and specifically under the isothermal condition, has simple and convenient operation and does not use expensive instruments and reagents; an amplification product can be developed directly by using a fluorescent dye and a result can be judged with naked eyes; the detection cost is low; and the invention is particularly suitable for small and medium size units and field tests.

Description

technical field [0001] The invention relates to a biological detection kit and method, in particular to a detection kit and method for Riemerella anatipestifer based on loop-mediated isothermal amplification technology. Background technique [0002] Riemerella anatipestifer (RA) is a Gram-negative, non-flagellated, non-spore-forming coryneform bacterium. The Riemerella anatipestifer disease caused by it is a contagious infectious disease that harms poultry and wild birds such as ducks, geese, and turkeys. It is also known as duck infectious serositis, new duck disease, duck sepsis, and duck plague syndrome disease and pasteurellosis anatipestifer, etc. The morbidity and mortality of the disease are usually 10 to 30%, but the mortality rate of sick ducks infected with Riemerella anatipestifer in some duck farms is as high as 75%. Once the Riemerella anatipestifer infection occurs in ducks and geese in a certain area, the disease will become an endemic disease, which is diff...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
Inventor 杨金龙付利芝杨睿杨松全程安春沈克飞杨柳
Owner CHONGQING ACAD OF ANIMAL SCI
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