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A pathogenic and fungal technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of unseen research reports on filamentous fungi, difficult cells, etc.
Active Publication Date: 2010-10-06
CHUGOKU IGAKU KAGAKUIN HIFUBIYOU KENKYUSHO
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However, because this method only uses thermal cracking to destroy the bacteria, it was most commonly used in bacteria in the past, while the outer layer of fungal cells has a thicker cell wall, which consists of a skeleton (fine fibers composed of insoluble polysaccharide crystals) and a matrix (polymer complex) Composition, the structure is very dense, and it is difficult to lyse cells simply by heat, so it is rare to use colony PCR technology for mycological experimental research, only sporadic applications for yeast, and no research for filamentous fungi Report
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Embodiment 1
[0027] 1. Colony culture:
[0028] Inoculate the filamentous fungi used in the experiment on the prepared PDA medium under aseptic conditions, and culture them at 28°C for 7-14 days. In bacterial normal saline, shake and vortex to disperse the mycelium mass as much as possible, take a small amount of prepared bacterial solution and inoculate it on the PDA plate medium, and incubate at 28°C for about 20-40h (depending on the growth of the colony). Colonies that were visible to the naked eye were considered experimental colonies.
[0029] 2. DNA template preparation method for colony PCR
[0030] Take a 0.2ml sterile PCR tube, add a small amount of high-pressure sterilized quartz sand, add 20μl sterile 1×TE buffer, and set aside. Scrape a small amount of the above-mentioned mycelium with a needle, add it to the prepared PCR tube, mark it, cover it tightly, boil it at 100°C for 5 minutes, then immediately put it in a -20°C refrigerator for 5 minutes, repeat twice, and put it in...
Embodiment 2
[0057] 1. Colony culture:
[0058] The fungus adopts the standard bacterial strain Aspergillus fumigatus, and the cultivation method is the same as that in Example 1.
[0059] 2. DNA template preparation method for colony PCR
[0060] With embodiment 1.
[0061] 3.PCR amplification
[0062] Multiplex PCR system primers used fungal universal primer NS, dermatophyte-specific primers and yeast-specific primers (see Table 2). PCR reaction system 25 μl, containing 4 μl 10× buffer, 1.5 μl MgCl 2 (10mmol / L), 2μl dNTPmix (10umol / L), 0.5μl each primer (10μmol / L), 2μl DNA template, 2U Taq DNA polymerase. The reaction conditions were 95°C for 5 minutes after pre-denaturation, 95°C for 1min, 52°C for 1min, 72°C for 1min, a total of 30 cycles, and finally 72°C for 10min. The amplified product was electrophoresed on 1.5% agarose gel, and 4 μl was applied to each sample well, the electrophoresis buffer was 1×TAE, the constant voltage was 80 V, and the electrophoresis was about 30 min. ...
Embodiment 3
[0064] Except that fungus adopts standard bacterial strain Trichophyton rubrum, all the other are with embodiment 2.
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Abstract
The invention relates to a fungus colony PCR method and a pathogenic fungus identification method. The fungus colony PCR method aims to achieve the high positive rate of amplification, and the pathogenic fungus identification method aims to achieve short detection time and reliable results. The fungus colony PCR method comprises the following steps of: inoculating fungus cultures onto a culture medium for culture; taking fungus colonies which begin to appear on the culture medium as experimentally available colonies; and preparing a DNA template for colony PCR amplification. The pathogenic fungus identification method comprises the following steps of: inoculating clinical sample cultures or pathogenic fungus cultures onto the culture medium for culture; taking the colonies which begin to appear on the culture medium as the experimentally available colonies; preparing the DNA template for the PCR amplification; and detecting amplification products. The fungus colony PCR method and the pathogenic fungus identification method have the advantages of improving the positive rate of the fungus colony PCR amplification, solving the problems of complex steps, wall-breakage difficulty, longtime and the like in fungus genomic DNA extraction, performing colony identification at the very beginning of the growth of pathogenic fungi, and achieving reliable results and identification time remarkably shorter than conventional fungus phenotype identification time.
Description
technical field [0001] The invention relates to a fungal colony PCR method and a method for identifying pathogenic fungi. Background technique [0002] At present, superficial fungal infection still maintains a high incidence and recurrence rate in the population, among which tinea capitis, onychomycosis, dermatophyte granuloma, recurrent candidal vaginitis, etc. still have diagnostic and therapeutic aspects. problem. Due to the continuous expansion of the immunocompromised population, the incidence of deep fungal infection is increasing rapidly year by year, and often threatens the life of the infected host. The success rate of its treatment depends on the early diagnosis and treatment of pathogenic bacteria. There are more than 300 kinds of pathogenic bacteria causing various fungal infections, of which more than 50 are common. The sensitivity of different fungal species to different antifungal drugs is quite different. For example, although Candida albicans and Aspergil...
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