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A method and kit for detecting trace fungi using single-cell sequencing

A cell and fungus technology, applied in the field of fungal detection kits, can solve the problems of difficult extraction of gDNA, fungal wall breaking, etc., and achieve the effects of good market prospects and considerable economic and social benefits.

Active Publication Date: 2018-12-04
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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  • A method and kit for detecting trace fungi using single-cell sequencing

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] 1. Obtaining a small amount of Cryptococcus gattii cells: microscopic smear, laser cutting, and obtaining a single target cell

[0068] 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). 2. Cell wall breaking of Cryptococcus gattii: Extraction of fungal protoplasts by combining chemical method and mixed enzyme method

[0069] 1) Add 200 μl of 0.8M D-sorbitol solution into the above centrifuge tube, soak the cells at 4°C for 2 hours;

[0070] 2) Prepare 100...

Embodiment 2

[0088] 1. Obtaining a small amount of Candida Dublin cells: Microsmear, laser cutting, and obtaining a single target cell

[0089] 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).

[0090] 2. Cell wall breaking of Candida Dublin: Extraction of fungal protoplasts by combining chemical method and mixed enzyme method

[0091] 1) Add 200 μl of 0.8M D-sorbitol solution into the above centrifuge tube, soak the cells at 4°C for 2 hours;

[0092] 2) Prepare 100ml of composite pretreatment age...

Embodiment 3

[0109] 1. Acquisition of trace amounts of Malassezia cells: Microsmear, laser cutting, to obtain a single target cell

[0110] 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).

[0111] 2. Malassezia cell wall breaking: the combination of chemical method and mixed enzyme method to extract fungal protoplasts

[0112] 1) Add 200 μl of 0.8M D-sorbitol solution into the above centrifuge tube, soak the cells at 4°C for 2 hours;

[0113] 2) Prepare 100ml of composite pretreatment agent: m...

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Abstract

The invention relates to the field of microorganisms and molecular biology, in particular to a method for detecting trace fungi by single-cell sequencing and a fungal detection kit prepared by the method. The method of the present invention includes the acquisition of trace fungal cells, the extraction of fungal protoplasts by breaking fungal cell walls, the extraction and amplification of gDNA traces of fungal protoplasts, the construction of trace gDNA libraries, genome sequencing, bioinformatics analysis and comparison, and determination of the types of fungi detected, etc. step. The method of the invention realizes high-efficiency detection of trace fungi, and can be directly applied to the separation, detection, identification and in-depth study of genetic information of trace and difficult fungal samples or mixed samples. The fungus detection method and kit of the present invention 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 product detection, medical detection and the like.

Description

technical field [0001] The invention relates to the field of microorganisms and molecular biology, in particular to a method for detecting trace fungi by single-cell sequencing and a fungal detection kit prepared by the method. Background technique [0002] 1. Overview of existing single-cell sequencing technologies [0003] 1) At present, single-cell sequencing technology is mainly used for research on the development of human or mammalian embryonic cells and stem cells. Such cells have only cell membranes and no cell walls, so there is no need to break the walls. It is relatively easy to extract gDNA from trace cells. [0004] 2) It has been reported that single-cell sequencing technology is used for 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 breaking and DNA extrac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12Q1/04
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