Novel specific molecular targets for 16 different serotypes of salmonella and rapid detection method thereof

A technology for Salmonella and Salmonella enteritidis, which is applied in the field of microbiological testing, can solve the problems of no PCR method establishment report, and the report of PCR detection method for new molecular targets specific to serotype Salmonella has not been found, and achieves short detection time, simple result judgment, low cost effect

Active Publication Date: 2020-04-17
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are almost no research reports on PCR detection targets and primers for other serotypes. Therefore, there are no reports on the establishment of PCR methods for detection of other serotypes.
[0

Method used

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  • Novel specific molecular targets for 16 different serotypes of salmonella and rapid detection method thereof
  • Novel specific molecular targets for 16 different serotypes of salmonella and rapid detection method thereof
  • Novel specific molecular targets for 16 different serotypes of salmonella and rapid detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Mining of new molecular targets specific to different species of Salmonella

[0063] According to the GenBank database and the complete genome DNA sequences of different serotypes of Salmonella self-tested by our team, bioinformatics analysis was carried out; 16 different serotypes of Salmonella (Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Delphi, Salmonella Indiana, Argonne Salmonella, Salmonella infantis, Salmonella lyssen, Salmonella Brundenloop, Salmonella Vershaw, Salmonella Albany, Salmonella Hadar, Salmonella Wertfrieden, Salmonella London, Salmonella turkey, Salmonella Senftenberg, and Salmonella Pomona ) specific gene fragment, the nucleotide sequence of said gene fragment is shown in SEQ ID NO.1-SEQ ID NO.72.

[0064] Wherein, the sequences SEQ ID NO.1-4 are used to identify the Salmonella enteritidis, the sequence SEQ ID NO.5 is used to identify the Salmonella typhimurium, and the sequences SEQ ID NO.6-13 are used to identify the Salm...

Embodiment 2

[0065] Example 2 Rapid Detection of Primer Effectiveness

[0066] 1) Primer design

[0067] According to the sequence SEQ ID NO.1-72 in Example 1, a specific PCR amplification primer set (including a forward primer and a reverse primer) was designed. The sequence of the primer set is shown in Table 1.

[0068] Table 1 Specific PCR detection primer set

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]2) the method for identifying said 16 different serotypes of Salmonella, the steps are as follows:

[0076] S1 DNA template preparation: Use a commercial bacterial genomic DNA extraction kit to extract the bacterial genomic DNA to be tested as the template DNA to be tested;

[0077] S2 PCR amplification: use one of the primer sets 1-72 to perform PCR amplification on the DNA of the sample to be tested;

[0078] ①PCR detection system:

[0079]

[0080]

[0081] in:

[0082] The primer sets 1-4 are used to identify the Salmonella enteritidis,...

Embodiment 3

[0095] The specificity evaluation of embodiment 3 Salmonella enteritidis PCR detection method

[0096] Take 91 strains of Salmonella enteritidis, 41 strains of Salmonella of non-target serotypes, and 22 strains of non-Salmonella, and perform PCR detection according to the method in Example 2. Wherein, the S1 DNA template is prepared by extracting the genomic DNA of each bacterium; during the S2PCR amplification, the primers used are any one of the primer sets 1-4.

[0097] The gel results of PCR amplification products are as follows: Figure 1~4 (The first and last ones in the gel graph of Salmonella Enteritidis are DNA markers, and the penultimate one is a negative control; the first and second ones in the gel graphs of other serotypes of Salmonella and non-Salmonella are DNA markers and positive controls, respectively) .

[0098] The strains and test results are shown in Table 2 below; in the table, "+" in the test result column means positive, and "-" means negative.

[...

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Abstract

The invention discloses specific novel molecular targets of 16 different serotypes of salmonella and a rapid detection method of the specific novel molecular targets. The invention provides a method for detecting salmonella enteritidis, which is used for different serotypes of salmonella. The invention method provides 72 specific molecular detection targets for different serotypes of salmonella including salmonella enteritidis, salmonella typhimurium, salmonella derby, salmonella indiana, salmonella agona, salmonella infantis, salmonella rissen, salmonella braenderup, salmonella virehow , salmonella albany, salmonella hadar, salmonella weltevreden, salmonella London, salmonella meleagridis, salmonella senftenberg and salmonella pomona, and corresponding PCR primer groups and a rapid detection method. The method provided by the invention has the advantages of short detection time, low cost, simple operation and strong specificity; meanwhile, the detection method can be used for detecting some strains with unexpressed antigens, avoiding the defects of immunodetection, and is more accurate in detection result, simpler in judgment and higher in practicability.

Description

technical field [0001] The invention belongs to the technical field of microbiological testing, and relates to a method for distinguishing Salmonella, in particular to 16 different serotype-specific new molecular targets of Salmonella and a rapid detection method thereof. Background technique [0002] Salmonella is one of the most common foodborne pathogens and one of the four major pathogens of diarrheal diseases worldwide. The initial symptoms of human Salmonella infection are nausea, vomiting, diarrhea, fever, etc. If not treated in time, the mortality rate is as high as 10%. Under normal circumstances, Salmonella can cause human infection if the consumption exceeds 105 CFU, and 15-20 CFU can cause Salmonella infection in highly susceptible people (Kokkinos et al., 2014). The severity of the disease depends on host factors and the serotype of Salmonella (WHO, 2018). [0003] The serotypes of Salmonella are complex and widely distributed. Almost most of the serotypes can...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/42
CPCC12Q1/689C12Q1/686C12Q2600/16Y02A50/30
Inventor 叶青华吴清平张菊梅尚玉婷杨小鹃陈谋通王涓丁郁张淑红薛亮吴浩明
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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