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Application of imp gene of Riemeria anatipestifer and its PCR detection kit and method

A detection kit technology for Riemerella anatipestifer, which is applied in the field of livestock and poultry detection, can solve the problems of E.

Active Publication Date: 2021-07-06
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 conducive to timely diagnosis of the cause, inspection of the pathogen and control of the spread of the disease
Aiming at these characteristics of Riemerella anatipestifer, many researchers have established some detection techniques, such as fluorescent antibody technique, ELLSA, immunohistochemistry, etc., which have been reported successively, but in practice, they all have different degrees of limitations, such as the need to target different serum type of antibody as a detection reagent

Method used

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  • Application of imp gene of Riemeria anatipestifer and its PCR detection kit and method
  • Application of imp gene of Riemeria anatipestifer and its PCR detection kit and method
  • Application of imp gene of Riemeria anatipestifer and its PCR detection kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, the establishment of Riemeria anatipestifer PCR method

[0019] Design of primer pairs for specific amplification of R. anatipestifer: The Imp gene was found from the genomic DNA sequence of R. anatipestifer through bioinformatics analysis. Imp is highly conserved in other Gram-negative bacteria and has multiple 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. It is used as the detection target gene of Riemerella anatipestifer, and the gene sequence is shown in SEQ ID NO.1.

[0020] (1) Primer design

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

Embodiment 2

[0031] Embodiment 2, PCR detection method specificity evaluation of Riemeria anatipestifer

[0032] According to the DNA template extraction method and PCR detection method in Implementation 1, Escherichia coli, Pasteurella, Salmonella, and various serotypes of Riemeria anatipestifer strains (reserved strains or serological tests identified as Riemerella anatipestifer The clinical isolates, as shown in Table 1) were tested.

[0033] Table 1. The strains used for specificity evaluation and the detection results of PCR test

[0034] Strain number Deposit number or clinically isolated and identified strains Strain name and serotype PCR result RA CH-1 (RCH) CCTCC NO.M2017702 Riemerella anatipestifer serotype 1 + RA CH-2 Identification of serotyped clinical isolates Riemerella anatipestifer serotype 2 + RCAD0166 Identification of serotyped clinical isolates Riemerella anatipestifer serotype 2 + RA ATCC11845 ATCC11845 Riemerella...

Embodiment 3

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

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

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Abstract

The present invention relates to a kind of PCR detection kit based on Riemerella anatipestifer Imp gene and method thereof, said kit contains the primer pair shown in SEQ ID NO.2 and SEQ ID NO.3; The nucleotide sequence of the Mererella Imp gene is shown in SEQ ID NO.1; the present invention detects Riemerella anatipestifer by taking the Imp gene as the target gene, which has remarkable specificity and high sensitivity, and is Early detection of Riemerella anatipestifer is of great significance.

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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Patent Type & Authority Patents(China)
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