Specific molecular target containing staphylococcus aureus standard strains as well as detection and application thereof

A staphylococcus, molecular target technology, applied in the field of bioengineering, can solve the problems of slow dissolution, automatic counting of colonies that affect the appearance, and scattered on the surface of the plate, etc.

Active Publication Date: 2021-04-02
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Another example is that Chinese patent CN102140423 B adopts 0.1 to 10 parts by mass of water-soluble sugar, 0.1 to 5 parts by mass of skimmed milk powder, 0.1 to 20 parts by mass of gelatin, and 0 to 10 parts by mass of activated carbon as protective agents for quantitative preservation. The protective agent does not mention the level of freeze-drying survival rate, and because the protective agent contains a certain concentration of gelatin, its solubility is affected at room temperature or when used in winter, and the dissolution rate is slow; in addition, the protective agent contains activated carbon. , Activated carbon is insoluble in aqueous solution, sinks to the bottom of the bottle after freeze-drying, and is scattered on the surface of the plate when it is recovered, affecting the appearance and automatic counting of colonies

Method used

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  • Specific molecular target containing staphylococcus aureus standard strains as well as detection and application thereof
  • Specific molecular target containing staphylococcus aureus standard strains as well as detection and application thereof
  • Specific molecular target containing staphylococcus aureus standard strains as well as detection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Isolation, identification and cultivation of embodiment 1 Staphylococcus aureus bacterial strain

[0037] Qualitative and quantitative tests are carried out on the collected samples at the same time, and the test methods are slightly adjusted on the basis of the national standard "Food Microbiological Inspection" GB4789.10-2010. Take 25g (mL) of the sample and add it to a sterile homogeneous bag of 225mL normal saline and shake it evenly to make a 1:10 sample solution, and take 1mL from it and add it to 9mL of 10% sodium chloride tryptone soybean broth In a test tube, a sample solution of 1:100 was prepared, and a sample solution of 1:1000 was prepared according to the above method; each gradient had 3 parallels, and the sample solution was placed in a constant temperature incubator at 37°C for 48 hours, and each The sample solutions with gradient concentrations were streaked on the culture medium of Staphylococcus aureus chromogenic plate, and cultured at 37°C for 24-4...

Embodiment 2

[0043] Physiological and biochemical characteristics and drug sensitivity characteristics of embodiment 2 Staphylococcus aureus strains

[0044] Staining microscopic examination: smear suspicious colonies, perform Gram staining, and observe the morphology under the microscope. Staphylococcus aureus is Gram-positive, and it forms a prototype grape bunch under the microscope ( figure 2 ).

[0045] Plasma coagulation enzyme test: Inoculate a single colony into 5mL BHI culture medium and incubate at 37°C for 18-24h. Aspirate 1 mL of the culture solution, add it to the plasma coagulase, and incubate at 37°C. After 2.5 hours, observe whether it coagulates every hour. If it does not coagulate after 6 hours, cultivate overnight and then observe and verify.

[0046] API Staph identification: scrape a single pink colony from the chromogenic plate, prepare a cell suspension with appropriate turbidity with physiological saline, and use API Staph biochemical identification reagent stri...

Embodiment 3

[0050] Molecular genetic background analysis of embodiment 3 Staphylococcus aureus bacterial strains

[0051] Multilocus sequence typing (MLST), staphylococcal protein A (SPA) typing, SCCmec typing and related gene detection were carried out on Staphylococcus aureus strains respectively.

[0052] (1) Experimental method

[0053] MLST typing is mainly based on the sequence analysis of seven S. aureus housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqil). Primers and amplification methods refer to previous literature reports. The 7 pairs of primers used were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (see Table 3 for primer sequences). PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, and 35 cycles; finally, extension at 72°C for 10 min. The reaction system (25 μL) contained: 12.5 μl 2×DreamTaq mastermix, 8.5 μl ultrapure water, 80...

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Abstract

The invention relates to the technical field of bioengineering, in particular to four staphylococcus aureus standard strains which have independent intellectual property rights in China and meet the domestic propagation rule. The staphylococcus aureus standard strains can be used as standard reference strains in different fields of foods, medicines, clinical examination and the like. The preservation numbers of the strains are GDMCC 60841, GDMCC 60843, GDMCC 60844 and GDMCC 60845 respectively. The four staphylococcus aureus strains have standard staphylococcus microscopic morphology and physiological and biochemical characteristics, the genetic background is clear, and information of drug resistance, virulence genes and molecular detection targets is clear. The invention also relates to agroup of specific target genes for detecting and identifying the four staphylococcus aureus standard strains and corresponding PCR primers. Finally, the invention further provides a staphylococcus aureus freeze-drying protective agent with high survival rate and specificity, and the staphylococcus aureus freeze-drying protective agent can be used for long-term storage of the standard strains.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a standard strain of Staphylococcus aureus containing a specific molecular target and its detection and application. Background technique [0002] Staphylococcus aureus (S.aureus) is one of the important food-borne pathogens that cause bacterial food poisoning. In my country, 20-25% of bacterial food poisoning cases are caused by Staphylococcus aureus. . Staphylococcus aureus can produce a variety of toxins, some toxins can withstand high temperature, and the human body can cause food poisoning after ingestion of a few micrograms; some toxins can cause damage to human white blood cells and macrophages, and some can cause fibrosis in the blood Protein deposition can lead to various infections such as toxic shock syndrome, osteomyelitis, suppurative endocarditis, bacteremia and fatal pneumonia in severe cases, threatening human health. Understanding the spread rules and pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/14C12N15/11C12N1/20C12N1/04C12R1/445
CPCC12Q1/689C12N1/04Y02A50/30
Inventor 吴诗吴清平代京莎丁郁陈谋通张菊梅薛亮王涓叶青华曾海燕古其会杨小鹃张淑红韦献虎张友雄庞锐卢勉飞张峰黄嘉慧陈鲁
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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