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Novel specific molecular targets for four common pathogenic staphylococci and rapid detection method thereof

A technology for staphylococcus and staphylococcus epidermidis, which is applied in the field of microbial testing, can solve the problems of less hemolytic staphylococcus and human staphylococcus, difficult to meet the needs of actual testing, false positive results and misjudgment, etc., and achieves short testing time and low cost. , the result is easy to determine the effect

Active Publication Date: 2020-05-15
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In practical applications, it has been found that although there are many target genes reported for Staphylococcus aureus and Staphylococcus epidermidis, they have high homology within the genus, and their specificity is poor, which can easily lead to false positive results and misjudgment
For example, the specificity verification of the primers designed for the nuc gene of Staphylococcus aureus using 17 species of staphylococci already in the laboratory found that the amplification results of Staphylococcus squirrel and Staphylococcus ludeng were positive and could not be identified by this target
However, Staphylococcus hemolyticus and Staphylococcus hominis are very few in the existing reporter gene targets, and it is difficult to meet the needs of actual detection
With the continuous discovery of new species of Staphylococcus during clinical treatment, the accurate identification based on the original molecular targets of Staphylococcus is facing great challenges

Method used

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  • Novel specific molecular targets for four common pathogenic staphylococci and rapid detection method thereof
  • Novel specific molecular targets for four common pathogenic staphylococci and rapid detection method thereof
  • Novel specific molecular targets for four common pathogenic staphylococci and rapid detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Discovery of specific new molecular targets for 4 common pathogenic staphylococci

[0048] Based on the GenBank database and the team’s self-tested staphylococcal genome DNA sequence, bioinformatics analysis; screening of 4 common pathogenic staphylococci (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus and Staphylococcus human) Specific gene fragments, the nucleotide sequences of the gene fragments are shown in SEQ ID NO. 1-19.

[0049] Wherein, the sequence SEQ ID NO. 1 to 5 is used to identify Staphylococcus aureus; the sequence SEQ ID NO. 6 to 10 is used to identify Staphylococcus epidermidis; the sequence SEQ ID NO. 11 to 15 is used to identify Staphylococcus haemolyticus; the sequence of SEQ ID NO. 16-19 is used to identify human Staphylococcus.

Embodiment 2

[0050] The fast detection method of Staphylococcus described in Example 2

[0051] 1) Primer design

[0052] According to the sequence SEQ ID NO. 1-19 in Example 1, a specific PCR amplification primer set (including forward primer and reverse primer) was designed. The sequence of the primer set is shown in Table 1 below.

[0053] Table 1 Specific PCR detection primer set

[0054]

[0055]

[0056] 2) The method of identifying Staphylococcus, the steps are as follows:

[0057] S1 DNA template preparation: the test strains are respectively enriched and cultured in LB liquid medium, and the bacterial genomic DNA is extracted separately using a commercial bacterial genomic DNA extraction kit as the test template;

[0058] S2 PCR amplification: use one of the primer sets 1-19 to perform PCR amplification on the DNA of the test sample

[0059] ①PCR detection system:

[0060]

[0061] among them:

[0062] When the template DNA is used to identify the presence of Staphylococcus aureus, use any one ...

Embodiment 3

[0074] Example 3 Specific evaluation of PCR detection method for Staphylococcus aureus

[0075] Take 91 strains of Staphylococcus aureus, 16 strains of non-target Staphylococcus (Staphylococcus epidermidis, Staphylococcus human, Staphylococcus hemolyticus, Staphylococcus ludunensis, etc.), 90 strains of non-Staphylococcus (Salmonella, Escherichia coli, Shiga) Spp.), PCR detection was carried out according to the method of Example 2. Among them, the S1 DNA template is prepared by separately extracting the genomic DNA of each bacteria; during S2 PCR amplification, the primer used is any one of primer sets 1 to 5. Set a blank control. The template of the blank control is an aqueous solution without genome.

[0076] The strains and test results of each bacteria used are shown in Table 2 below. In the table, the "+" in the test result column indicates positive and "-" indicates negative. The PCR product electrophoresis results are as figure 1 As shown; among them, (a) 1~91 are 91 Stap...

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Abstract

The invention discloses novel specific molecular targets for four common pathogenic staphylococci and a rapid detection method thereof. Nineteen novel specific molecular detection targets for identifying four common pathogenic staphylococci-staphylococcus aureus, staphylococcu sepidermidis, staphylococcus haemolyticus and staphylococcus hominis, and corresponding PCR primer sets and a PCR rapid detection method are provided. The detection method can be used for detecting some strains with insensitive biochemical reaction, so that the defects of biochemical identification can be made up, and practicability can be enhanced; and the detection method also has the advantages of being simple in operation, easy in result determination, short in detection time, strong in specificity and low in cost.

Description

Technical field [0001] The invention belongs to the technical field of microbial testing, relates to a method for identifying staphylococci, and specifically relates to four common pathogenic staphylococcus-specific new molecular targets and rapid detection methods thereof. Background technique [0002] Staphylococci is one of the most common nosocomial pathogens, and it is also an important pathogen directly related to animal (dairy cow) mastitis. Staphylococcus aureus, as a model bacteria in the genus Staphylococcus, is one of the four major food-borne pathogens. Studies have shown that food poisoning incidents caused by Staphylococcus aureus account for 25% of food-borne microbial food poisoning incidents each year, and the enterotoxins produced by it can cause vomiting and diarrhea and cause major harm to humans. According to the "National Food Safety Standard Limits of Pathogenic Bacteria in Food" (GB 29921-2013), the concentration of Staphylococcus aureus in the sample sha...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12N15/11
CPCC12Q1/689C12Q1/686C12Q2565/125Y02A50/30
Inventor 吴清平周宝青叶青华吴诗张菊梅丁郁薛亮王涓陈谋通李滢韦献虎庞锐
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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