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Multiple PCR (polymerase chain reaction) method for identifying salmonella enteritidis, salmonella typhimurium, salmonella pullorum and salmonella gallinarum

A technology of Salmonella enteritidis and Salmonella, which is applied in the field of multiple PCR detection of different serotypes of Salmonella, can solve the problems of fast base mutation and unsuitable bacterial identification, and shorten the diagnosis time

Inactive Publication Date: 2013-06-05
YANGZHOU UNIV
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Problems solved by technology

Although 23S rRNA has a large molecule and a lot of information, the base mutation rate is fast, and it is also not suitable for bacterial identification

Method used

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  • Multiple PCR (polymerase chain reaction) method for identifying salmonella enteritidis, salmonella typhimurium, salmonella pullorum and salmonella gallinarum
  • Multiple PCR (polymerase chain reaction) method for identifying salmonella enteritidis, salmonella typhimurium, salmonella pullorum and salmonella gallinarum
  • Multiple PCR (polymerase chain reaction) method for identifying salmonella enteritidis, salmonella typhimurium, salmonella pullorum and salmonella gallinarum

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Embodiment Construction

[0020] Concrete technical scheme of the present invention is as follows:

[0021] 1. Primer Design

[0022] According to the purpose of the experiment, after homology comparison, a pair of primers were designed according to the specific gene hut of Salmonella. In addition, according to Salmonella enteritidis (SdfⅠ), Salmonella typhimurium (SPY), Salmonella pullorum (glgc), Salmonella gallinarum typhi (glgc A pair of primers were designed respectively for the serotype-specific genes of Spec and Spec). The primer sequences are as follows:

[0023]

[0024] Note: Merged base code R=A / G Y=C / T.

[0025] 2. Bacterial DNA Extraction

[0026] (1) Pick a single colony and shake it overnight in 3ml liquid LB medium. Use a 1.5ml centrifuge tube to take 400μL of the bacterial solution, centrifuge at 12000rpm for 5min, discard the supernatant, resuspend in 200μL of sterile ultrapure water, and centrifuge at 12000rpm for 5min. Discard the supernatant, resuspend with 100 μL sterile ul...

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Abstract

The invention relates to a multiple PCR (polymerase chain reaction) method for identifying salmonella enteritidis, salmonella typhimurium, salmonella pullorum and salmonella gallinarum. The multi-PCR method comprises the following steps of: with the extracted DNA (deoxyribonucleic acid) of a bacterium to be detected as a template, carrying out mPCR (multiple polymerase chain reaction) amplification on five pairs of primers as shown in SEQ ID NO.1-10; and carrying out agarose gel electrophoresis on mPCR amplification products, and then determining according to electrophoresis results. According to the multiple PCR method, the synthesized primers are used for carrying out PCR amplification, a diseased material can be directly ground and amplified by adopting the established multiple PCR, different salmonella serotypes can be detected in one system, serum glass plate agglutination is completed in only about six hours compared with three days for the traditional serum glass plate agglutination after separation pure culture in clinical detection, the diagnosis time is greatly shortened, and a basis is provided for clinical diagnosis.

Description

technical field [0001] The invention relates to a method for detecting different serotypes of Salmonella by multiplex PCR, and the serotypes mainly involved are Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum and Salmonella gallinarum typhi. Background technique [0002] Salmonella (Salmonella) is a general term for a group of common bacteria, some of which only cause disease in humans, some only cause disease in animals, and some are pathogenic to both humans and animals. Salmonellosis is a general term for different forms of diseases caused by various types of Salmonella to humans, domestic animals and wild animals. [0003] Pigs, poultry, dogs, and cats infected with Salmonella can cause symptoms such as gastroenteritis, abortion, and sepsis. With the expansion of intensive animal breeding and the widespread use of antibacterial drugs in clinics and animal feeds, the drug resistance of Salmonella is becoming more and more serious, and the infection ra...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/42
Inventor 陈素娟彭大新杨林魏荣陈继明刘秀梵
Owner YANGZHOU UNIV
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