Method for collecting fungal spores in deep fungal infection specimen

A technology of deep fungi and fungal spores, applied in the biological field, can solve the problems of low positive rate, poor timeliness, poor fungal antigen and antibody detection specificity, etc., and achieve the effect of improving the positive rate and efficient collection.

Inactive Publication Date: 2020-09-01
杭州同创医学检验实验室有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Among the current means of etiological diagnosis of fungal infection, the positive rate of histomorphological staining and imaging examination is low, the specificity of fungal antigen and antibody detection is poor, and gene amplification technology is limited by factors such as specimens, amplification primers, and amplification conditions. The results are often unstable, and the routine fungal culture in the microbiology room takes a long time and has poor timeliness, which is difficult to meet the needs of early clinical diagnosis and treatment

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  • Method for collecting fungal spores in deep fungal infection specimen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] like figure 1 As shown, the method for collecting fungal spores in deep fungal infected tissues comprises the following steps:

[0023] (a) Pretreatment: take samples from deeply infected tissues, with a volume of 1×1×2cm, and put the samples into a pre-cooled sterile petri dish; in a biological safety cabinet, use 12cm sterile surgical scissors Crush the tissue and grind it repeatedly in a 2mL glass grinder under ice bath conditions; filter the ground tissue mixture through a 300-mesh nylon mesh, this operation can filter cell membranes and a large number of red blood cells to ensure the collection of prokaryotic or eukaryotic pathogens; After the filtration is completed, use 0.01mol / LPBS solution to wash and filter repeatedly, and transfer to a sterile 20mL centrifuge tube, add 5mL red blood cell lysate (ACKLysisBuffer) to the centrifuge tube, the red blood cell lysate can be removed without damaging the nucleated cells Red blood cells; then centrifuge at 4°C and 150...

Embodiment 2

[0028] like figure 1 As shown, the method for collecting fungal spores in deep fungal infection body fluids comprises the following steps:

[0029] (a) Treatment of body fluids with deep fungal infection: 3 ml of blood was collected, centrifuged at 4000 rpm for 15 min, and the concentrated fungal mixture was collected.

[0030] (b) Fungal culture: the mixture was transferred to a fungal blood culture bottle using a No. 7 cell needle, and conventional fungal culture was performed using a blood culture instrument.

[0031] (c) Collecting fungal spores: Take 5ml of the positive mixture in the fungal blood culture bottle and add it to a 20mL sterile centrifuge tube, centrifuge at 5000rpm for 5min to obtain a precipitate, and then transfer the precipitate to the built-in tube. The fungal spores were collected in a 2 mL sterile EP tube with a pore size of 10 μm biofiltration membrane and centrifuged at 14000 rpm for 10 min at high speed.

Embodiment 3

[0033] like figure 1 As shown, the method for collecting fungal spores from alveolar lavage fluid includes the following steps:

[0034] (a) Treatment of alveolar lavage fluid: take 20 μL of alveolar lavage fluid as a cell smear, and select qualified specimens by microscope observation. The qualified specimens are 2% of total epithelial cells and 2% of red blood cells. The ratio is 6%; filter the qualified specimen through a 300-mesh nylon mesh to remove exfoliated epithelial cells and mucus, and collect all alveolar lavage fluid containing fungi into a 20ml centrifuge tube; centrifuge at 4°C, 1500rpm for 15 minutes, Discard the supernatant and collect the fungus-containing mixture.

[0035] (b) Fungal culture: the mixture was transferred to a fungal blood culture bottle using a No. 7 cell needle, and conventional fungal culture was performed using a blood culture instrument.

[0036] (c) Collect fungal spores: Take 5ml of the mixture in the fungal blood culture bottle and a...

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Abstract

The invention discloses a method for collecting fungal spores in a deep fungal infection specimen, and belongs to the technical field of biology. The method comprises the following steps: (a) pretreatment: treating a deep fungal infection specimen, and collecting a mixture containing fungi and metabolites; (b) fungus culture: transferring the mixture into a fungus blood culture bottle by using a cell needle, and carrying out conventional fungus culture by using a blood culture instrument; and (c) collecting fungal spores: centrifuging the positive mixture in the fungal blood culture bottle, discarding the supernatant, transferring the precipitate into a 2 mL sterile EP tube internally provided with a biological filter membrane with a pore diameter of 10 [mu]m, centrifuging at a high speed,filtering, and collecting the fungal spores. Compared with a conventional method, the method has the advantages that pathogenic bacteria infected by invasive fungi can be quickly, efficiently and accurately collected, and the positive rate of culture is increased.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method for collecting fungal spores from deep fungal infection specimens. Background technique [0002] Fungi are widely distributed microorganisms in the natural environment and are also common commensal bacteria on human skin and mucous membranes. However, when the immune system of the body is damaged or the functional barrier is damaged, the fungus will invade the mucous membrane, internal organs and deep tissues. Under certain conditions, it can spread and cause systemic infection, resulting in fungemia and deep tissue infection. There is a higher fatality rate. There are 1.5 million species of fungi discovered so far, about 500 of which are pathogenic bacteria. In the past 30 years, due to the increase in patients with malignant tumors, AIDS, diabetes, and organ transplantation, these patients have low immunity, long-term use of broad-spectrum antibiotics...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N3/00C12N1/14
CPCC12N1/14C12N3/00
Inventor 李玉串胡伟华余嘉伟包秀玲
Owner 杭州同创医学检验实验室有限公司
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