Standard strain of Yersinia enterocolitica containing specific molecular targets and its detection and application

A technology for Yersinia and enterocolitis, applied in the field of microbiological testing, can solve the problems of not reflecting the contamination and transmission of the bacteria well, achieve clear virulence genes and drug resistance spectrum, biotype and serum well-defined effect

Active Publication Date: 2022-05-20
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

Most of these standard strains are derived from foreign clinical isolates, which cannot well reflect the contamination and spread of the bacteria in food.
At the same time, due to the different types of the main epidemic strains of Yersinia enterocolitica in food in different countries and regions, the characteristics of the main epidemic strains abroad cannot be used as the representative of the characteristics of the main epidemic strains in China.

Method used

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  • Standard strain of Yersinia enterocolitica containing specific molecular targets and its detection and application
  • Standard strain of Yersinia enterocolitica containing specific molecular targets and its detection and application
  • Standard strain of Yersinia enterocolitica containing specific molecular targets and its detection and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Isolation and cultivation of Yersinia enterocolitica bacterial strain

[0033] The collected food samples were thoroughly shredded under aseptic conditions, 25g of the sample was weighed and diluted 10 times, then enriched with PBS, cultured by shaking at 22°C-25°C for 2-3 days or standing for 5 days. After 20 seconds of alkali treatment (0.5 mL enrichment solution + 4.5 mL KOH), inoculate on CIN agar and incubate at 30°C for 24-48 hours. Pick five typical colonies and inoculate them on nutrient agar, and culture them at 30°C for 24 hours. Under sterile conditions, add the bacterial solution into a glycerol tube with a final concentration of 25%, and store in a -80°C refrigerator. Purified colonies are ready for subsequent screening experiments as well as biochemical identification and biotyping.

Embodiment 2

[0034] Example 2 Physiological and biochemical characteristics and serotype analysis of Yersinia enterocolitica standard strain

[0035] Modified Kirschner's double sugar test: Pick 3 to 5 suspicious colonies isolated and inoculate them on modified Kirschner's double sugar iron agar respectively. When inoculating, first draw a line on the slope, and then puncture the bottom layer, at 26°C±1°C After culturing for 24 hours, further biochemical identification was carried out on the cultures whose slopes and bottoms turned yellow and did not produce gas.

[0036] Colony morphology: using improved Y plate culture, the colony morphology of Yersinia enterocolitica standard strain is a round, pink colony with bile precipitation rings on the edge (results such as Figure 1-3 ).

[0037] Urease test and kinetic observation: Pick up a full ring of suspicious cultures obtained from the modified Klebsonian disaccharide test with an inoculation loop and inoculate them into urea medium. ...

Embodiment 3

[0048] Example 3 Carrying characteristics of virulence factors of Yersinia enterocolitica standard strain

[0049] The virulence genes carried by the strains were identified by PCR. The primers used were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (see Table 4 for primer sequences). PCR amplification system (25 μL) includes: 2×Ferment PCR mix, 12.5 μL; 0.4 μM upstream and downstream primers; ddH 2 O, 8.5 μL and genomic DNA, 2 μL. 8-10 μL of PCR products were loaded on 1.5% agarose gel for electrophoresis separation (130V, 35 min), using 2000pb DNA Marker.

[0050] Table 4: Primer sequences and amplified fragments used in the detection of Yersinia enterocolitica virulence genes.

[0051]

[0052]

[0053] The virulence genes carried by the standard strain of Yersinia enterocolitica are as follows:

[0054] The virulence genes carried by the standard strain GDMCC 60852 are: ystB-ymoA-fes-sat-inv-hrep-fepA-fepD-myfA.

[0055] The virulence genes carried by...

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Abstract

The present invention provides three standard strains that can be used as controls for Yersinia enterocolitica (Yersinia enterocolitica) related experiments. The standard strains have typical Yersinia enterocolitica colony morphology and physiological and biochemical characteristics, It can be used to test the accuracy of Yersinia enterocolitica selective medium, and can be used as a control sample in the detection of common physiological and biochemical phenotypes and molecular characteristics of Yersinia enterocolitica; the standard strain contains For the corresponding specific molecular target (such as the nucleotide sequence shown in SEQ ID NO: 1-11), specific PCR amplification primers containing the specificity of the molecular target are also provided. The present invention also provides a freeze-drying protective agent for Yersinia enterocolitica, the freeze-drying survival rate of the protective agent can reach more than 85%; storage at -20°C can guarantee its order of magnitude for at least one year The above does not change, so it can be used for long-term storage of quality control strains.

Description

technical field [0001] The invention belongs to the technical field of microbial inspection, and in particular relates to a standard bacterial strain of Yersinia enterocolitica; physiological, biochemical and molecular characteristics of the bacterial strain; corresponding molecular targets and PCR amplification primers and a preservation of Yersinia enterocolitica method. Background technique [0002] Yersinia enterocolitica (Y.enterocolitica), as a Gram-negative zoonotic pathogen, is widely distributed in nature and is the main cause of Yersinia. Because of its ability to grow in a low temperature environment (0-4°C), frozen and refrigerated food has become a new important source of infection of the disease in recent years. The main symptoms of Yersin's disease are gastroenteritis, pseudoappendicitis and diarrhea, etc., and a small number of patients will develop chronic diseases such as reactive arthritis due to infection. The monitoring data of the European Food Safety...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12N1/20C12N1/04C12R1/01
CPCC12Q1/689C12N1/04Y02A50/30
Inventor 王涓吴清平丁郁陈惠元张菊梅陈谋通薛亮吴诗叶青华代京莎徐环庞锐曾海燕雷涛古其会韦献虎张友雄张淑红杨小鹃刘鸣王惠贤陈鲁
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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