Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Specific PCR detection method of Riemerella anatipestifer

A technology of Riemer's anatipestifer and a detection method is applied in the field of specific PCR detection of Riemerella anatipestifer, which can solve problems such as unfavorable diagnosis of etiology, investigation of pathogenic conditions, economic loss of duck breeding industry, harm to duck breeding industry, and the like. To achieve the effect of simple result judgment, short detection time, and reduced detection cost

Active Publication Date: 2014-07-09
SICHUAN AGRI UNIV
View PDF1 Cites 0 Cited by
  • 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. It is one of the most serious diseases that endanger the duck industry and has caused 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, but there are deficiencies in the identification of Riemerella anatipestifer with phenotypic indicators.
In addition, the traditional identification method is complicated to operate, time-consuming and labor-intensive, which is not conducive to timely diagnosis of the cause, investigation of the cause 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., but they all have different degrees of limitations in practice.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Specific PCR detection method of Riemerella anatipestifer
  • Specific PCR detection method of Riemerella anatipestifer
  • Specific PCR detection method of Riemerella anatipestifer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Establishment of PCR method for Riemerella anatipestifer

[0016] Through bioinformatics analysis, the gyrB gene, which plays an important role in the process of specific DNA transcription, replication, recombination and repair, was found from the genomic DNA sequence of Riemerella anatipestifer, and it was used as the detection target gene of Riemerella anatipestifer. The sequence is shown in SEQ ID NO: 1;

[0017] The DNA sequence of this gene is imported in the primer design software Primer Premier 5.0 to design primers, the setting GC% range is 40-60%, and the product size range is 150-300bp, selects primers from the alternative primer pair, and the primer sequences are as follows ( Primers were synthesized by Shanghai Yingjun Technology Service Co., Ltd.):

[0018] GYRB-L: 5'-AGAGCGAGAAGAAAAAACCT-3' (SEQ ID NO: 2);

[0019] GYRB-R: 5'-CTCCCATAAGCATAGAGAAGA-3' (SEQ ID NO: 3);

[0020] Step 2, preparation of DNA template

[0021] Inoculate the strains of various ...

Embodiment 2

[0028] PCR specificity evaluation experiment

[0029] According to the DNA template extraction method and PCR detection method in Implementation 1, Escherichia coli, Salmonella, Staphylococcus aureus, Pasteurella multocida, Bacillus pumilus, Streptococcus, Salmonella typhimurium, Salmonella Dublin, Salmonella pullorum, Standard strains such as Riemerella anatipestifer (as shown in Table 1, the strains shown in the table are all obtained by those skilled in the art through open channels) were subjected to amplification reactions.

[0030] For the test results of the specificity test, see figure 1 , please refer to the accompanying drawings for negative control strains and strain numbers. In the picture: Lanes 1-10: Pasteurella.multiocida PM966, Salmonella CMCC 50083-4, Staphylococcus aureus ATCC 6538, Escherichia 046, Streptococcus CMCC32223, Bacillus pumilus subtil CMCC63202 strain, Salmonella.typhimurium 50115-13 strain, Salmonella.dulin 50761 strain, Salmonella pullorum 50...

Embodiment 3

[0035] PCR sensitivity evaluation experiment

[0036] The Riemerella anatipestifer template DNA obtained by extracting in Example 1 was measured by OD260 / 280, and the concentration of the total DNA solution of Riemerella anatipestifer was 350 ng / μl, and was diluted 10 times with sterile water, and co-diluted 6 gradients, 1 μL of each gradient was added to the PCR reaction system, the amplified product was detected by gel electrophoresis, and the gel electrophoresis results were observed in a gel imager; figure 2As shown in the figure: lanes 1-6: reactions with DNA template contents of 350ng, 35ng, ​​3.5ng, 350pg, 35pg, 3.5pg and 350fg respectively; lane 7: ddH 2 O; Lane M: DL2000bp molecular weight standard. 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 35 pg, which has good sensitivity.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a PCR detection method of Riemerella anatipestifer. The detection method comprises the following steps: 1, amplification primers are designed according to a conserved sequence of gyrB gene in a Riemerella anatipestifer genomic DNA sequence, wherein the conserved sequence is represented by SEQ ID NO:1; 2, DNA is extracted from a sample and is subjected to PCR amplification; 3, the obtained amplified product is detected through gel electrophoresis to determine whether the sample contains Riemerella anatipestifer; 5, if there is a corresponding single amplified strip in the electrophoresis result, the sample contains Riemerella anatipestifer; and 6, if there is no corresponding single amplified strip in the electrophoresis result, the sample contains no Riemerella anatipestifer. By adopting the detection method of the invention to detect Riemerella anatipestifer, the detection time is short, the cost is low, the detection result is specific, and the result determination is simple.

Description

technical field [0001] The invention relates to a detection method in the field of common poultry diseases, in particular to a specific PCR detection method for Riemerella anatipestifer. Background technique [0002] Riemerella anatipestifer (RA) is the pathogenic bacterium of duck infectious serositis, which is one of the most important diseases that endanger waterfowl farming, especially duck farming. Among poultry, ducks are the most susceptible to infection, and many varieties of ducks such as Cherry Valley Duck, Muscovy Duck, Shelduck Duck, and Lijia Duck can be affected. 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. It is one of the most serious di...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
Inventor 程安春王雪平汪铭书朱德康陈孝跃
Owner SICHUAN AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com