Application of riemerella anatipestifer Imp gene, PCR detection kit and method thereof

A Riemerella anatipestifer and detection kit technology, which is applied in the field of livestock and poultry detection, can solve the problems of unfavorable diagnosis of etiology, inspection of pathogenic conditions, confusion of Escherichia coli and Salmonella, and economic losses in the duck industry. The effect of simple result judgment and reduced testing cost

Active Publication Date: 2018-11-16
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease is prevalent in almost all duck raising areas in the world, causing huge economic losses to the duck industry
The traditional identification of RA usually detects phenotypic indicators such as its cultural characteristics, morphological staining, physiological and biochemical characteristics, and certain chemical compositions of the bacteria. to confuse
In addition, the traditional identification method is complicated to operate, time-consuming and laborious, which is not con

Method used

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  • Application of riemerella anatipestifer Imp gene, PCR detection kit and method thereof
  • Application of riemerella anatipestifer Imp gene, PCR detection kit and method thereof
  • Application of riemerella anatipestifer Imp gene, PCR detection kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0018] Example 1. Establishment of PCR method for Riemerella anatipestifer

[0019] Design a primer pair that specifically amplifies Riemerella anatipestifer: The Imp gene is found from the genomic DNA sequence of Riemerella anatipestifer through bioinformatics analysis. Imp is highly conserved among other Gram-negative bacteria and has a variety of Important biological functions, in addition to participating in the process of lipopolysaccharide export and assembly to the outer membrane, are also related to the formation of bacterial envelope and the tolerance of bacterial organic solvents. This is used as the target gene for detection of Riemerella anatipestifer, and the gene sequence is shown in SEQ ID NO.1.

[0020] (1) Primer design

[0021] Input the DNA sequence of the gene into the primer design software Primer Premier 5.0 to design primers, set the product size range to 100-500bp, select the primers from the candidate primer pairs, and the primer sequences are as follows (pr...

Example Embodiment

[0031] Example 2: Evaluation of specificity of PCR detection method for Riemerella anatipestifer

[0032] According to the DNA template extraction method and PCR detection method in implementation 1, the strains of Escherichia coli, Pasteurella, Salmonella, and multiple serotypes of Riemerella anatipestifer (preserved strains or serological tests were identified as Riemerella anatipestifer) The clinically isolated strains, as shown in Table 1) were tested.

[0033] Table 1. Strains used for specificity evaluation and PCR test results

[0034] Strain number

[0035] In Table 1, -: PCR result is negative; +: PCR result is positive.

[0036] See the results of specific experiments figure 1 ,From figure 1 It can be seen that, except Riemerella anatipestifer of various serotypes, all the other strains have no specific amplified band at 445bp.

Example Embodiment

[0037] Example 3. Sensitivity evaluation experiment of PCR detection method for Riemerella anatipestifer

[0038] The R. anatipestifer template DNA extracted in Example 1 was determined by OD260 / 280, and the concentration of the R. anatipestifer total DNA solution was 150ng / μl, which was diluted by 10 times with sterile water and co-dilution There are 8 gradients, and 2 μL of each gradient is added to the PCR reaction system, and the DNA template is subjected to PCR amplification detection according to the method in step (3) of Example 1. Take 5μL of the PCR amplification product, electrophoresis in a 1% agarose gel, observe the gel electrophoresis result in the gel imager as figure 2 Shown. by figure 2 It can be seen that a clear band can be seen in the fifth lane, and the corresponding DNA concentration is 15pg, which has extremely high sensitivity.

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Abstract

The invention relates to a PCR detection kit based on riemerella anatipestifer Imp gene and a method thereof. The kit contains primer pairs shown as SEQ ID NO.2 and SEQ ID NO.3, wherein the nucleotidesequence of the riemerella anatipestifer Imp gene is shown as SEQ ID NO.1. The detection kit and method provided by the invention adopt Imp gene as the target gene to detect riemerella anatipestifer,have significant specificity and high sensitivity, and are of important significance for early detection of riemerella anatipestifer.

Description

technical field [0001] The invention belongs to the field of livestock and poultry detection, relates to the application of the Riemerella anatipestifer Imp gene, and also relates to a PCR detection kit based on the Riemerella anatipestifer Imp gene and a corresponding detection method. Background technique [0002] Riemerella anatipestifer (RA) is the pathogenic bacterium of duck infectious serositis, which is one of the important diseases that endanger the duck industry. The disease is an acute or chronic sepsis process, characterized by fibrinous pericarditis, perihepatitis, air sac inflammation, meningitis, and some caseous salpingitis and arthritis. The morbidity rate is 10%-90%, and the case fatality rate is as high as 80%, which can occur throughout the year. The disease is prevalent in almost all duck raising areas in the world, causing huge economic losses to the duck industry. The traditional identification of RA usually detects phenotypic indicators such as its ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 汪铭书邱浩程安春
Owner SICHUAN AGRI UNIV
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