Multi-PCR (Polymerase Chain Reaction) specific primer for detecting bacterial enteritis pathogens of pig and application thereof

A bacterial enteritis and specific technology, applied in the field of molecular biology, can solve the problems of target blindness, time-consuming, large amount of reagents, etc., and achieve the effect of high sensitivity and strong specificity

Inactive Publication Date: 2017-05-31
WENS FOODSTUFF GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cultivation process takes a long time and the target is blind, especially the anaerobic cultivation requires high hardware and operational proficiency, and the PCR identification of a single pathogen needs to detect three pathogens separately, which takes a long time and consumes a lot of reagents

Method used

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  • Multi-PCR (Polymerase Chain Reaction) specific primer for detecting bacterial enteritis pathogens of pig and application thereof
  • Multi-PCR (Polymerase Chain Reaction) specific primer for detecting bacterial enteritis pathogens of pig and application thereof
  • Multi-PCR (Polymerase Chain Reaction) specific primer for detecting bacterial enteritis pathogens of pig and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Design of primers

[0038] The research results of ASIMK.BEJ et al. showed that the gene encoding glucosidase uidA can only be detected in Escherichia coli but not in other intestinal bacteria. Rahn et al. found that the Salmonella invA gene is widely distributed in various serotypes of Salmonella. This gene can encode the surface protein of adsorption and invasion of epithelial cells. This protein is related to the pathogenicity of Salmonella and is distributed in almost all known Salmonella. Not found in non-Salmonella species. The exotoxin secreted by Clostridium perfringens is its pathogenic factor. According to the different types of exotoxin produced, it can be divided into five serotypes: A, B, C, D and E. Among them, α-toxin (C. perfringens alpha toxin (CPA) is a common virulence factor produced by various types of Clostridium perfringens, which can destroy the integrity of the cell membrane and cause cell lysis, thus having the characteristics of cytotoxici...

Embodiment 2

[0051] Example 2 specificity test

[0052] With the presence of Escherichia coli, Salmonella and Clostridium perfringens CVCC12-57 culture fluid as positive controls, the culture fluid of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Streptococcus suis, and Pasteurella suis Carry out specific detection, use the method and primer of embodiment 1, all fail to amplify any band except positive control, positive disease material expands 166bp band, 449bp band and 579bp respectively through PCR, see figure 2 , indicating that the primers and detection method of the present invention can verify and identify whether the sample contains Escherichia coli, Salmonella and Clostridium perfringens.

Embodiment 3

[0053] Embodiment 3 Sensitivity test

[0054] The Escherichia coli, Salmonella and Clostridium perfringens CVCC12-57 strain DNA extracted in Example 1 were respectively made 10 times of gradient dilution with sterilized double distilled water, i.e. 10 9 CFU, 10 8 CFU, 10 7 CFU, 10 6 CFU, 10 5 CFU, 10 4 CFU content of E. coli, Salmonella and 10 9 CFU, 10 8 CFU, 10 7 CFU, 10 6 CFU, 10 5 CFU, 10 4 CFU, 10 3 The Clostridium perfringens DNA of the CFU content is used as a template, and each dilution takes 1 μ L as a template, and according to the method in Example 1, each pathogen is detected for a single pathogen and the simultaneous presence of three pathogens, and a negative control is set. Observe the results of the positive bands, and calculate the sensitivity based on the highest dilution of the template used for the positive expected bands. The results show that the minimum detection concentrations of single pathogenic Escherichia coli, Salmonella and Clostridium ...

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Abstract

The invention relates to the field of molecular biology and production of animal medicine, in particular to a multi-PCR (Polymerase Chain Reaction) specific primer for detecting bacterial enteritis pathogens, such as escherichia coli, salmonellae and clostridium perfringens, of a pig; nucleotide sequences are shown as SEQ ID NO:1 to SEQ ID NO:6; the specificity, the repeatability and the sensitivity are high. The invention also discloses a method which is established on the basis of the primer and is used for detecting the bacterial enteritis pathogens, such as the escherichia coli, the salmonellae and the clostridium perfringens, of the pig; the enteritis of the pig which is caused by single infection or mixed infection of the escherichia coli, the salmonellae and the clostridium perfringens can be distinguished quickly; the primer can be used to identify and diagnose quickly and effectively, and is beneficial to formulating targeted prevention and control measures to perform immune prevention and control on the bacterial enteritis pathogens of the pig in clinical parturition and ensure smooth parturition of the raised pig.

Description

technical field [0001] The invention relates to the field of molecular biology of veterinary medicine, in particular to multiple specific PCR primers for detecting porcine bacterial enteritis pathogenic Escherichia coli, Salmonella and Clostridium perfringens and applications thereof. Background technique [0002] Porcine bacterial enteritis persists in clinical production for a long time and leads to slow growth and mass death. The pathogens causing porcine enteritis are mainly Escherichia coli, Salmonella, and Clostridium perfringens, among which Escherichia coli causes yellow and white scour in piglets, diarrhea and intestinal edema in weaned piglets, etc.; Salmonella causes paratyphoid fever in piglets and diarrhea in fattening pigs, etc.; Clostridium membranes cause red diarrhea in piglets and intestinal flatulence in fattening pigs. Porcine bacterial enteritis has a short course of disease, rapid spread, rapid development of the disease, and high mortality. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12Q1/04C12N15/11C12R1/19C12R1/42C12R1/145
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 王雷宋延华潘永飞
Owner WENS FOODSTUFF GRP CO LTD
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