Target genes for detecting salmonella paratyphi, PCR primer pair as well as detection method and applications thereof

A technology for paratyphoid C and Salmonella, applied in botany equipment and methods, microbial-based methods, biochemical equipment and methods, etc., can solve problems such as long detection cycle, inaccurate detection results, and complicated detection process, and achieve The effect of shortened detection time, strong singleness, and simple result judgment

Active Publication Date: 2015-05-13
NANJING AGRICULTURAL UNIVERSITY
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  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to obtain the specific detection target gene of Salmonella paratyphi C through genome comparison analysis, thereby carry out PCR amplification and electrophoresis detection of the target gene

Method used

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  • Target genes for detecting salmonella paratyphi, PCR primer pair as well as detection method and applications thereof
  • Target genes for detecting salmonella paratyphi, PCR primer pair as well as detection method and applications thereof
  • Target genes for detecting salmonella paratyphi, PCR primer pair as well as detection method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Specificity evaluation of different bacterial species by PCR detection method

[0033] 1. Screening of serotype-specific genes of Salmonella paratyphi C

[0034] The complete genome sequence of Salmonella paratyphi C RKS4594 strain (NC_012125.1) was obtained in the NCBI database. According to the method of comparing genomes, each gene of the genome of the bacteria was compared using the Blast program of the NCBA website, and the serum was selected. The genes with high type homology (E value=0, Query cover=0%) and low homology with other microorganisms (Query cover<10%) were used as quasi-specific genes of Salmonella paratyphi C. A total of 7 quasi-specific genes of Salmonella paratyphi C were obtained by this method, and multiple pairs of primers were designed for each gene, and the specificity was verified by using other serotypes of Salmonella preserved in the laboratory. According to the PCR results, the SPC_0871, SPC_0872 and SPC_0908 genes were determined as the s...

Embodiment 2

[0047] Sensitivity Evaluation of PCR Detection Method to Different Template Concentrations

[0048] The screening and primer design steps of Salmonella paratyphi C serotype-specific genes are as in Example 1.

[0049] Preparation of DNA template: Pick a single colony of Salmonella paratyphi C and transfer it to 30 ml of LB liquid medium, and cultivate overnight at 37°C. The bacterial genomic DNA mini-extraction kit produced by Shanghai Sangon Bioengineering Technology Service Co., Ltd. was used to extract the total DNA of Salmonella paratyphi C, and the concentration was determined to be 98.33ng / ul. Make a 10-fold gradient dilution with sterile water, and dilute a total of 6 gradients as a PCR template.

[0050] Establishment of PCR reaction system: establishment of PCR detection system: 25ul reaction system includes: 2×Master 12.5ul, 5uM primer pair 1ul, template 5ul, supplemented with ddH 2 0 to 25ul. The reaction program of PCR was designed: pre-denaturation at 94°C for ...

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Abstract

The invention provides three specific target genes for detecting salmonella paratyphi, a corresponding PCR primer pair and a method for detecting the target genes by using the primer pairs. The PCR primer pair is designed according to the three specific target genes for detecting salmonella paratyphi, the specific target genes are high in stability and strong in specificity, and through reasonably optimizing a PCR system, and adopting gel electrophoresis detection means, salmonella paratyphi can be quickly and accurately identified. The detection method provided by the invention comprises the following steps: (1) extracting the DNA of a sample, and carrying out PCR amplification; (2) detecting an amplified product through gel electrophoresis; and (3) comparing rear electrophoretic bands, if the bands exist at 523 bp, 212 bp and 169 bp positions, proving that the sample contains salmonella paratyphi. According to experimental comparison and analysis, the method disclosed by the invention has the characteristics of strong singularity, reliable detection results, and simple result determination, and can be widely applied in the field of food sanitation.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to a pair of PCR primers for detecting Salmonella paratyphi C, a specific target gene, a detection method using the pair of primers and the target gene, a kit including the pair of primers and the primer For the application of the kit for detecting Salmonella paratyphi C. Background technique [0002] Salmonella paratyphi C (Salmonella paratyphi C) is one of the main Salmonella serotypes that cause human infection, and the main host of this serotype strain is humans. Infected people will have different degrees of malignancy, vomiting, diarrhea and fever, and severe cases will have sepsis and so on. In a recent research report, it was shown that Salmonella paratyphi C can infect healthy human body through certain foods through the oral cavity, thus causing the above-mentioned symptoms in various aspects. Zhu Xiang and Bao Yunjuan published "A Food Poisonin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12N15/11C12N15/31C12R1/42
CPCC12Q1/686C12Q2565/125
Inventor 别小妹翟立公陆兆新张充吕凤霞
Owner NANJING AGRICULTURAL UNIVERSITY
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