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Method for detecting trace fungus by using single-cell sequencing and kit

A single-cell sequencing and fungal technology, applied in the field of microorganisms and molecular biology, can solve the problems of difficult extraction of gDNA, fungal wall breaking, etc., and achieve the effect of good market prospects and considerable economic and social benefits.

Active Publication Date: 2016-09-07
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] The problem to be solved by the present invention is: the purpose of the present invention is to overcome the deficiencies in the existing trace fungal detection technology, such as being difficult to effectively break the wall of trace fungi that cannot be cultivated or difficult to cultivate; it is difficult to extract a sufficient amount of trace fungi gDNA that can be used to build a gene library; it is difficult to detect specific fungi in complex samples, etc.

Method used

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  • Method for detecting trace fungus by using single-cell sequencing and kit

Examples

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Effect test

Embodiment 1

[0059] 1. Obtaining a small amount of Cryptococcus gattii cells: microscopic smear, laser cutting, and obtaining a single target cell

[0060] Apply a small amount of sample to a laser microdissection coated glass slide (Leica Membrane Slides PEN2.0 micron or similar product), use a laser microdissection system (Leica LMD7000 or similar product) to find the target cells under 63 times magnification, and use The laser intensity is 40 microjoules (the laser power range can be set to 40±3), and the selected single fungal cells are laser microdissected, and they are dropped into a sterile centrifuge tube. Repeat the above operation to obtain a total of 10 fungal cells (can be obtained Cell number range 1-100).

[0061] 2. Cell wall breaking of Cryptococcus gattii: Extraction of fungal protoplasts by combining chemical method and mixed enzyme method

[0062] 1) Add 200 μl of 0.8M D-sorbitol solution into the above centrifuge tube, soak the cells at 4°C for 2 hours;

[0063] 2) Pr...

Embodiment 2

[0081] 1. Obtaining a small amount of Candida Dublin cells: Microsmear, laser cutting, and obtaining a single target cell

[0082] Apply a small amount of sample to a laser microdissection coated glass slide (Leica Membrane Slides PEN2.0 micron or similar product), use a laser microdissection system (Leica LMD7000 or similar product) to find the target cells under 63 times magnification, and use The laser intensity is 43 microfocus (the laser power can be set to 40 ± 3) to perform laser microdissection on the selected single fungal cell, and it falls into a sterile centrifuge tube to obtain a total of 1 fungal cell (the range of cell number can be obtained 1-100 pieces).

[0083] 2. Cell wall breaking of Candida Dublin: Extraction of fungal protoplasts by combining chemical method and mixed enzyme method

[0084] 1) Add 200 μl of 0.8M D-sorbitol solution into the above centrifuge tube, soak the cells at 4°C for 2 hours;

[0085] 2) Prepare 100ml of composite pretreatment age...

Embodiment 3

[0102] 1. Acquisition of trace amounts of Malassezia cells: Microsmear, laser cutting, to obtain a single target cell

[0103] Apply a small amount of sample to a laser microdissection coated glass slide (Leica Membrane Slides PEN2.0 micron or similar product), use a laser microdissection system (Leica LMD7000 or similar product) to find the target cells under 63 times magnification, and use The laser intensity is 38 μJ (can be set to laser power 40±3) to perform laser microdissection on the selected single fungal cells, and drop them into sterile centrifuge tubes. Repeat the above operation to obtain a total of 60 fungal cells (can be obtained Cell number range 1-100).

[0104] 2. Malassezia cell wall breaking: the combination of chemical method and mixed enzyme method to extract fungal protoplasts

[0105] 1) Add 200 μl of 0.8M D-sorbitol solution into the above centrifuge tube, soak the cells at 4°C for 2 hours;

[0106] 2) Prepare 100ml of composite pretreatment agent: m...

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Abstract

The invention relates to the field of microorganism and molecular biology, and in particular relates to a method for detecting trace fungus by using single-cell sequencing and a fungus detection kit produced by using the method. The method comprises the steps of extraction of trace fungus cells, extraction of fungus protoplasm by fragmentation of fungus cell wall, extraction and amplification of trace fungus protoplasm gDNA, database creation of gDNA, genome sequencing, bioinformatic analysis comparison, and judgment on the type of the detected fungus. The method disclosed by the invention realizes effective detection on trace fungus, and can be directly applied to separation, detection and identification of trace difficult fungus samples or mixed samples and deep study of genetic information. The fungus detection method and the kit are applicable to the fields of industrial production, environmental monitoring, air detection, soil detection, water quality detection, food detection, drug detection, cosmetic detection, health care products detection and medical detection.

Description

Technical field [0001] The invention relates to the field of microorganisms and molecular biology, and in particular to a method for detecting trace amounts of fungi using single-cell sequencing and a fungus detection kit made using the method. Background technique [0002] 1. Overview of existing single-cell sequencing technologies [0003] 1) Currently, single-cell sequencing technology is mainly used for research on the development of human or mammalian embryonic cells and stem cells. Such cells only have cell membranes and no cell walls, so there is no need to break the walls. It is easier to extract gDNA from trace amounts of cells. [0004] 2) It has been reported that single-cell sequencing technology is used in cell sequencing research on plant seed development and plant endophytic fungi. Such cells can be isolated and cultured, so it is relatively easy to obtain pure cell lines for molecular detection experiments. [0005] 2. Overview of existing fungal wall-breaki...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6806C12Q1/6869C12Q2535/122C12Q2537/165C12Q2527/125C12Q2531/113C12Q1/04C12Q1/6895C12R2001/645C12N1/145C12N15/10
Inventor 杨英王升启周喆王澎李珍李宗玮
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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