WO2018006458A1 - 一种利用单细胞测序检测微量真菌的方法及试剂盒 - Google Patents

一种利用单细胞测序检测微量真菌的方法及试剂盒 Download PDF

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WO2018006458A1
WO2018006458A1 PCT/CN2016/093734 CN2016093734W WO2018006458A1 WO 2018006458 A1 WO2018006458 A1 WO 2018006458A1 CN 2016093734 W CN2016093734 W CN 2016093734W WO 2018006458 A1 WO2018006458 A1 WO 2018006458A1
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cells
fungal
centrifuge tube
treatment agent
testing
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杨英
王升启
周喆
王澎
李珍
李宗玮
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中国人民解放军军事医学科学院放射与辐射医学研究所
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Definitions

  • 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.
  • single-cell sequencing technology is mainly used for human or mammalian embryonic cells and stem cell development research.
  • Such cells have only cell membranes and no cell wall, so there is no need to break the wall, and it is easier to extract gDNA from trace cells.
  • Enzymatic method snail enzyme, cellulase, lysozyme, lytic enzyme, etc., the efficiency is low, and the extracted DNA is not enough for the amount of the sample to be built.
  • PCR and gene chip technology suitable for a range of fungal detection, targeted primers and probes are not designed, and are not suitable for completely unknown species.
  • One-generation sequencing after PCR amplification using universal primers It is suitable for single unknown fungal detection, generally used for culturable pure bacteria detection, and is not suitable for one-time detection of complex samples.
  • next-generation metagenomic sequencing it can be used for the detection of complex samples, but because the fungal DNA is difficult to extract compared with other microorganisms or mammals, it will generate a large amount of data irrelevant to the target species, and the data is difficult to identify. .
  • the method of the invention can completely overcome the above technical defects by performing the steps of breaking the wall, extracting and amplifying the gDNA, and performing the next generation sequencing library establishment and genome sequencing analysis, thereby realizing the high-efficiency detection of the micro fungus.
  • the method of the invention can be directly applied to the separation, detection, identification and genetic information of trace, difficult fungal samples or mixed samples.
  • the fungal detection kit made according to the method of the invention can be widely used in various fields such as industrial production, environmental monitoring, air detection, soil testing, water quality testing, food testing, drug testing, cosmetics testing, health product testing and medical testing.
  • the promotion and application of the technology of the present invention will have a good market prospect and generate considerable economic and social benefits.
  • the problem to be solved by the present invention is that the object of the present invention is to overcome the deficiencies of the existing micro-fungal detection technology, such as difficulty in efficiently breaking the micro-organisms which are difficult to culture or culture, and difficult to extract sufficient amounts of micro-fungals.
  • the gDNA that can be used for genome building; it is difficult to detect specific fungi in complex samples.
  • the invention aims to establish a complete micro-fungal detection technical solution to solve the technical problem that it is difficult to separate and culture, and it is difficult to carry out molecular detection and genetic information research on micro-fungals in complex samples by conventional methods.
  • the method of the invention obtains and extracts protoplasts from a single fungal cell in a complex sample, in particular, efficiently removes the fungal cell wall, and performs gDNA extraction, amplification and database construction on the fungal protoplast, and is sequenced with a new generation.
  • the technique combines the analysis of genomic information, rather than the determination and analysis of single or multiple specific sequences at the traditional PCR or gene chip level, ultimately enabling accurate detection of the fungus.
  • the present invention provides a method for detecting trace fungi using single cell sequencing, comprising the following steps:
  • microfungal cells A micro fungus sample was applied to a laser microdissection slide glass slide, and the target cells were found by laser microdissection system, and the selected single fungus was selected by laser intensity of 37-43 microjoules. The cells are subjected to laser microdissection to obtain a single target cell, and the above operation is repeated to obtain a total of 1-100 cells of interest;
  • Micro-gDNA database construction the above-mentioned fungal gDNA is constructed by using a DNA building kit
  • Genomic sequencing whole genome sequencing of the fungal gDNA constructed above;
  • Bioinformatics analysis The data obtained by sequencing is assembled by using relevant software, and the assembly result is compared with the related public database to determine the type of fungus detected.
  • the fungi detectable by the method of the present invention include Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, Candida dublin, Candida; Aspergillus fumigatus, yellow in Aspergillus Aspergillus, Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, Aspergillus fumigatus; Cryptococcus neoformans, Cryptococcus cryptus, and variants of the above species; the above-mentioned various species and their varieties belong to clinical medicine Although the bacteria can be cultured in vitro, it is difficult to be isolated and cultured successfully in real clinical samples due to factors such as drug use by patients.
  • the fungus detectable by the method of the present invention further includes Malassezia, which belongs to a harsh fungus, which is harsh on culture conditions, and needs to be supplemented with special nutrients in the culture medium to be cultured successfully.
  • the fungi detectable by the method of the invention further include histoplasma, imonium, S. skrjab, horse Penicillium fuliginea, Paraspora turf, dermatitis buds, Coccidioides; the above species are difficult to culture successfully in vitro.
  • the fungal cell wall is broken in step (2) of the method of the invention: the protoplast is extracted by chemical method and mixed enzymatic method, wherein the mixed enzyme treatment agent is snail enzyme 6 mg/mL and lysing enzyme 4 mg. /mL; After adding the mixed enzyme treatment agent to the centrifuge tube, it was treated at 37 ° C for 6 hours; finally, 4 ⁇ l of the cell suspension was retained.
  • the mixed enzyme treatment agent is snail enzyme 6 mg/mL and lysing enzyme 4 mg. /mL
  • the fungal cell wall is broken in the step (2) in the method of the invention: the protoplast is extracted by chemical method and mixed enzymatic method, wherein the mixed enzyme treatment agent is snail enzyme 6 mg/mL and lysozyme 4 mg. /mL; After adding the mixed enzyme treatment agent to the centrifuge tube, it was treated at 45 ° C for 10 hours; finally, 5 ⁇ l of the cell suspension was retained.
  • the mixed enzyme treatment agent is snail enzyme 6 mg/mL and lysozyme 4 mg. /mL
  • the fungal cell wall is broken in step (2) of the method of the present invention: the protoplast is extracted by chemical method and mixed enzymatic method, wherein the mixed enzyme treatment agent is snail enzyme 4 mg/mL, lytic enzyme 4 mg/mL and lysozyme 4 mg/mL; after adding the mixed enzyme treatment agent to the centrifuge tube, the treatment was carried out at 35 ° C for 8 hours; finally, 6 ⁇ l of the cell suspension was retained.
  • the mixed enzyme treatment agent is snail enzyme 4 mg/mL, lytic enzyme 4 mg/mL and lysozyme 4 mg/mL
  • the fungal cell wall is broken in step (2) of the method of the present invention: the protoplast is extracted by chemical method and mixed enzymatic method, wherein the mixed enzyme treatment agent is snail enzyme 4 mg/mL, lytic enzyme 3 mg/mL and cellulase 3 mg/mL; after adding a mixed enzyme treatment agent to the centrifuge tube, it was treated at 37 ° C for 3 hours; finally, 6 ⁇ l of the cell suspension was retained.
  • the mixed enzyme treatment agent is snail enzyme 4 mg/mL, lytic enzyme 3 mg/mL and cellulase 3 mg/mL
  • the fungal cell wall is broken in the step (2) of the method of the present invention: the protoplast is extracted by chemical method and mixed enzymatic method, wherein the mixed enzyme treatment agent is snail enzyme 4 mg/mL and lysozyme 3 mg. /mL and cellulase 3 mg/mL; after adding the mixed enzyme treatment agent to the centrifuge tube, the treatment was carried out at 37 ° C for 12 hours; finally, 8 ⁇ l of the cell suspension was retained.
  • the mixed enzyme treatment agent is snail enzyme 4 mg/mL and lysozyme 3 mg. /mL and cellulase 3 mg/mL
  • the fungal cell wall is broken in the step (2) in the method of the invention: the protoplast is extracted by chemical method and mixed enzymatic method, wherein the mixed enzyme treatment agent is lysing enzyme 5 mg/mL, lysozyme 4 mg/mL and cellulase 3 mg/mL; after adding the mixed enzyme treatment agent to the centrifuge tube, the treatment was carried out at 37 ° C for 6 hours; finally, 10 ⁇ l of the cell suspension was retained.
  • the mixed enzyme treatment agent is lysing enzyme 5 mg/mL, lysozyme 4 mg/mL and cellulase 3 mg/mL
  • the fungal cell wall is broken in step (2) of the method of the present invention: the protoplast is extracted by chemical method and mixed enzymatic method, wherein the mixed enzyme treatment agent is snail enzyme 4 mg/mL, lytic enzyme 2 mg/mL, lysozyme 3 mg/mL, and cellulase 4 mg/mL; after adding the mixed enzyme treatment agent to the centrifuge tube, the treatment was carried out at 37 ° C for 6 hours; finally, 6 ⁇ l of the cell suspension was retained.
  • the mixed enzyme treatment agent is snail enzyme 4 mg/mL, lytic enzyme 2 mg/mL, lysozyme 3 mg/mL, and cellulase 4 mg/mL
  • the present invention also provides a fungal detection kit prepared according to the micro fungus detection method of the present invention, which can be applied to the following fields: industrial production, environmental monitoring, air detection, soil testing, water quality testing, Food testing, drug testing, cosmetic testing, health product testing, and medical testing.
  • the micro fungus detection method and corresponding kit of the invention overcome many shortcomings of the prior art, and provide a solution for separating and obtaining difficult fungi from complex samples; and providing an optimized technical solution for efficient wall breaking of micro fungi; It also provides a complete set of technical solutions for genome sequencing analysis of trace fungi in complex samples, and finally achieves efficient detection of trace fungi.
  • the method of the invention can be directly applied to the separation, detection, identification and in-depth study of genetic information of trace, difficult fungal samples or mixed samples.
  • the fungal detection kit prepared according to the method of the invention can be widely used in various fields such as industrial production, environmental monitoring, air detection, soil testing, water quality testing, food testing, drug testing, cosmetics testing, health product testing, and medical testing.
  • the popularization and application of the technology of the present invention will have a good market prospect and generate considerable economic and social benefits.
  • Figure 1 is an electrophoresis diagram of DNA quality control detection, in which: "M” is a DNA marker, No. 1-3 is a gDNA positive control that can be built, and No. 4-6 is a fungal single-cell gDNA extracted from the experiment. Sample and positive control The DNA has an obvious band of ⁇ 10kb.
  • step 2 Connect 4 ⁇ l of cell suspension in step 2, add 3 ⁇ l Buffer D2 in REPLI-g Single Cell Kit (Qiagen, Germany), mix and centrifuge instantaneously, incubate at 65 °C for 10 min; add 3 ⁇ l kit to Stop Solution, mix and instantaneously centrifuge Temporarily stored on ice; add 40 ⁇ l of PCR reaction solution to each amplification reaction (prepared according to the kit instructions: H 2 O sc 9 ⁇ l, REPLI-g sc Reaction Buffer 29 ⁇ l, REPLI-g sc DNA Polymerase 2 ⁇ l), 30 ° C The reaction was carried out for 8 hours; the REPLI-g DNA polymerase was inactivated at 65 ° C for 3 min, which is the extracted G. cryptococcus gDNA.
  • the DNA quality control test reached the second-generation sequencing database requirement, OD260/OD280: 1.82, DNA concentration: 75 ng/ ⁇ l, integrity > 10 kb, as shown in Figure 1.
  • M is the DNA marker
  • No. 1-3 is the gDNA positive control for the library
  • No. 4-6 is the fungal single-cell gDNA extracted from the experiment.
  • the DNA of the sample and the positive control has obvious ⁇ 10kb band.
  • the gDNA of the above library was subjected to whole genome sequencing using an Illumina MiSeq instrument and corresponding reagents (MiSeq 600cycles Reagent V3 Illumina, USA).
  • the sequencing data was assembled using SPAdes software, and the assembly result was compared with the NCBI public database nt library, and it was determined to be C. cryptus.
  • a small sample was applied to a laser microdissection slide glass slide (Leica Membrane Slides PEN 2.0 micron or similar), and the target cells were found at 63 times magnification using a laser microdissection system (Leica LMD7000 or similar).
  • the laser intensity is 43 microjoules (laser power 40 ⁇ 3).
  • the selected single fungal cells are laser microdissected and placed in a sterile centrifuge tube to obtain a total of 1 fungal cell. 1-100).
  • Candida cell wall breaking in Dublin chemical method combined with mixed enzyme method to extract fungal protoplast
  • the gDNA of the above library was subjected to whole genome sequencing using an Illumina MiSeq instrument and corresponding reagents (MiSeq 600cycles Reagent V3 Illumina, USA).
  • the sequencing data was assembled using SPAdes software, and the assembly results were compared with the NCBI public database nt library, which was judged to be Candida Dublin.
  • a small sample was applied to a laser microdissection slide glass slide (Leica Membrane Slides PEN 2.0 micron or similar), and the target cells were found at 63 times magnification using a laser microdissection system (Leica LMD7000 or similar).
  • the laser intensity is 38 microjoules (laser power 40 ⁇ 3).
  • the selected single fungal cells are laser microdissected and placed in a sterile centrifuge tube. A total of 60 fungal cells are obtained by repeating the above operation. The number of cells ranges from 1-100).
  • step 2 Connect 6 ⁇ l of cell suspension in step 2, add 3 ⁇ l Buffer D2 in REPLI-g Single Cell Kit (Qiagen, Germany), mix and centrifuge, incubate at 65 °C for 10 min; add 3 ⁇ l kit to Stop Solution, mix and instantaneously centrifuge Temporarily stored on ice; add 40 ⁇ l of PCR reaction solution to each amplification reaction (prepared according to the kit instructions: H 2 O sc 9 ⁇ l, REPLI-g sc Reaction Buffer 29 ⁇ l, REPLI-g sc DNA Polymerase 2 ⁇ l), 30 ° C The reaction was carried out for 8 hours; the REPLI-g DNA polymerase was inactivated at 65 ° C for 3 min, which is the extracted Malassezia gDNA.
  • the gDNA of the above library was subjected to whole genome sequencing using an Illumina MiSeq instrument and corresponding reagents (MiSeq 600cycles Reagent V3 Illumina, USA).
  • the sequencing data was assembled using SPAdes software, and the assembly result was compared with the NCBI public database nt library, and it was determined to be Malassezia.
  • micro-Immunia cells acquisition of micro-Immunia cells: microstaining, laser cutting, obtaining single target cells
  • a small sample was applied to a laser microdissection slide glass slide (Leica Membrane Slides PEN 2.0 micron or similar), and the target cells were found at 63 times magnification using a laser microdissection system (Leica LMD7000 or similar).
  • the laser intensity is 40 microjoules (laser power 40 ⁇ 3).
  • the selected single fungal cells are laser microdissected and placed in a sterile centrifuge tube. A total of 20 fungal cells are obtained by repeating the above operation. The number of cells ranges from 1-100).
  • Immune bacteria cell wall breaking chemical method combined with mixed enzyme method to extract fungal protoplast
  • the gDNA of the above library was subjected to whole genome sequencing using an Illumina MiSeq instrument and corresponding reagents (MiSeq 600cycles Reagent V3 Illumina, USA).
  • the sequencing data was assembled using SPAdes software, and the assembly result was compared with the NCBI public database nt library, and it was determined to be Imonobacter.
  • a small sample was applied to a laser microdissection slide glass slide (Leica Membrane Slides PEN 2.0 micron or similar), and the target cells were found at 63 times magnification using a laser microdissection system (Leica LMD7000 or similar).
  • the laser intensity is 37 microjoules (laser power 40 ⁇ 3).
  • the selected single fungal cells are laser microdissected and placed in a sterile centrifuge tube. A total of 100 fungal cells are obtained by repeating the above operation. The number of cells ranges from 1-100).
  • step 2 Connect 8 ⁇ l of cell suspension in step 2, add 3 ⁇ l Buffer D2 in REPLI-g Single Cell Kit (Qiagen, Germany), mix and centrifuge instantaneously, incubate at 65 °C for 10 min; add 3 ⁇ l kit to Stop Solution, mix and instantaneously centrifuge Temporarily stored on ice; add 40 ⁇ l PCR reaction solution to each amplification reaction (prepared according to the kit instructions: H 2 O sc 9 ⁇ l, REPLI-g sc Reaction Buffer 29 ⁇ l, REPLI-g sc DNA Polymerase 2 ⁇ l), 30 ° C The reaction was carried out for 8 hours; the REPLI-g DNA polymerase was inactivated at 65 ° C for 3 min, which was the extracted Aspergillus niger gDNA.
  • the gDNA of the above library was subjected to whole genome sequencing using an Illumina MiSeq instrument and corresponding reagents (MiSeq 600cycles Reagent V3 Illumina, USA).
  • the sequencing data was assembled using SPAdes software, and the assembly result was compared with the NCBI public database nt library, and it was determined to be Aspergillus niger.
  • a small sample was applied to a laser microdissection slide glass slide (Leica Membrane Slides PEN 2.0 micron or similar), and the target cells were found at 63 times magnification using a laser microdissection system (Leica LMD7000 or similar).
  • Laser intensity 42 microjoules (laser power 40 ⁇ 3) laser microdissection of selected single fungal cells, falling into sterile centrifuge tubes, and repeating the above operations to obtain 30 fungal cells (available The number of cells ranges from 1-100).
  • the gDNA of the above library was subjected to whole genome sequencing using an Illumina MiSeq instrument and corresponding reagents (MiSeq 600cycles Reagent V3 Illumina, USA).
  • the sequencing data was assembled using SPAdes software, and the assembly results were compared with the NCBI public database nt library, and it was determined to be dermatitis buds.
  • microsporum cells microstaining, laser cutting, obtaining single target cells
  • a small sample was applied to a laser microdissection slide glass slide (Leica Membrane Slides PEN 2.0 micron or similar), and the target cells were found at 63 times magnification using a laser microdissection system (Leica LMD7000 or similar).
  • the laser intensity is 40 microjoules (laser power 40 ⁇ 3).
  • the selected single fungal cells are laser microdissected and placed in a sterile centrifuge tube. Five fungal cells are obtained by repeating the above operation. The number of cells ranges from 1-100).
  • step 2 Connect 6 ⁇ l of cell suspension in step 2, add 3 ⁇ l Buffer D2 in REPLI-g Single Cell Kit (Qiagen, Germany), mix and centrifuge, incubate at 65 °C for 10 min; add 3 ⁇ l kit to Stop Solution, mix and instantaneously centrifuge Temporarily stored on ice; add 40 ⁇ l of PCR reaction solution to each amplification reaction (prepared according to the kit instructions: H 2 O sc 9 ⁇ l, REPLI-g sc Reaction Buffer 29 ⁇ l, REPLI-g sc DNA Polymerase 2 ⁇ l), 30 ° C The reaction was carried out for 8 hours; the REPLI-g DNA polymerase was inactivated at 65 ° C for 3 min, which is the extracted crude sporozoite gDNA.
  • the gDNA of the above library was subjected to whole genome sequencing using an Illumina MiSeq instrument and corresponding reagents (MiSeq 600cycles Reagent V3 Illumina, USA).
  • the sequencing data was assembled using SPAdes software, and the assembly result was compared with the NCBI public database nt library, and it was determined to be Coccidioides.

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Abstract

一种利用单细胞测序检测微量真菌的方法及利用该方法制成的真菌检测试剂盒。所述方法包括微量真菌细胞的获取、真菌细胞壁破壁提取真菌原生质、微量真菌原生质gDNA的提取和扩增、微量gDNA建库、基因组测序、生物信息学分析比对、判定所检测真菌的种类等步骤。所述方法实现了对微量真菌的高效检测，可以直接应用于微量、疑难真菌样本或混合样本的分离、检测、鉴别及遗传信息的深入研究。所述真菌检测方法及试剂盒适用于工业生产、环境监测、空气检测、土壤检测、水质检测、食品检测、药品检测、化妆品检测、保健品检测以及医学检测等领域。

Description

一种利用单细胞测序检测微量真菌的方法及试剂盒 技术领域
本发明涉及微生物及分子生物学领域，尤其涉及一种利用单细胞测序检测微量真菌的方法及利用该方法制成的真菌检测试剂盒。
背景技术
1.现有单细胞测序技术概述
1)目前单细胞测序技术主要用于人或哺乳动物胚胎细胞、干细胞发育研究，此类细胞只有细胞膜，没有细胞壁，因而无需破壁，从微量细胞中提取gDNA比较容易。
2)有报道单细胞测序技术用于植物种子发育、植物内生真菌的细胞测序研究，此类细胞可以被分离培养，所以比较容易获得纯细胞系进行分子检测实验。
2.现有的真菌破壁、DNA提取技术概述
1)物理方法：于研钵中加液氮研磨、研磨仪低温研磨，但此法需要大量的菌体，不能应用于微量样本。
2)化学方法：氯化苄提取法等，易降解，所提取的DNA完整性差，一般只够做PCR，难以达到用于基因组建文库的完整性要求。
3)酶法：蜗牛酶、纤维素酶、溶菌酶、溶壁酶等，效率低，所提取的DNA不够微量样本进行建库的量。
3.现有对微量真菌样本的检测技术概述
1)镜检：直接在显微镜下观察，虽然简便、快速，但灵敏度低，需要丰富经验，极易漏检。
2)培养：培养过程中真菌的形态和生化指标可作为检测的重要标准，但耗时长、易污染、很多真菌(尤其是病原真菌)难以在人工培养条件下培养成功。
3)PCR及基因芯片技术：适用于有明确范围的真菌检测，需要有针对性的设计引物、探针，不适用于完全未知的物种。
4)使用通用引物进行PCR扩增后进行一代测序：适用于单一的未知真菌检测，一般用于可培养的纯菌检测，不适用于复杂样本的一次性检测。
5)应用新一代宏基因组测序：可用于复杂样本的检测，但由于真菌的DNA与其他微生物或哺乳动物相比难以提取，所以会产生大量与目的菌种无关的数据，数据的甄别难度很大。
根据以上对现有技术的概述可以看出，目前针对微量真菌的检测技术主要存在以下技术缺点：
1.难以对微量、不可培养或培养难度大的真菌进行高效破壁。
2.难以对微量真菌提取足量的可进行基因组建库的gDNA。
3.难以对复杂样本中特定的真菌进行检测。
总而言之，真菌破壁提取完整基因组DNA一直是本领域中的难题，尤其是微量真菌样本，如何在微量的复杂样本中，提取可以达到二代测序建文库所需的真菌gDNA，并对其进行全基因组测序是目前还未有人报道的。
本发明方法通过取真菌微量细胞进行破壁，提取、扩增gDNA，并进行新一代测序的文库建立、基因组测序分析等步骤，可以很好的克服上述技术缺陷，从而实现对微量真菌的高效检测，本发明方法可以直接应用于微量、疑难真菌样本或混合样本的分离、检测、鉴别及遗传信息的深入研究。根据本发明方法制成的真菌检测试剂盒可广泛应用于工业生产、环境监测、空气检测、土壤检测、水质检测、食品检测、药品检测、化妆品检测、保健品检测以及医学检测等多种领域。本发明技术的推广应用将会具有良好的市场前景并产生可观的经济及社会效益。
发明内容
解决的技术问题
本发明需要解决的问题是：本发明的目的是克服现有微量真菌检测技术所存在的不足，如难以对微量、不可培养或培养难度大的真菌进行高效破壁；难以对微量真菌提取足量的可进行基因组建库的gDNA；难以对复杂样本中特定的真菌进行检测等问题。
技术方案
本发明旨在建立一套完整的微量真菌检测技术方案，以解决目前存在的难以分离培养、难以用常规方法对复杂样本中的微量真菌进行分子检测和遗传信息研究的技术难题。
本发明方法通过复杂样本中单个真菌细胞的分离获取和对其原生质的提取，尤其是对该真菌细胞壁的高效破除，以及对上述真菌原生质进行gDNA提取、扩增和建库，并与新一代测序技术结合分析基因组信息，而不是传统的PCR或基因芯片层次的单个或多个特定序列的测定和分析，最终实现对该真菌的准确检测。
首先，本发明提供了一种利用单细胞测序检测微量真菌的方法，包括以下步骤：
(1)微量真菌细胞的获取：将微量真菌样本涂于激光显微切割覆膜载玻片上，用激光显微切割系统找到目的细胞，用激光强度37-43微焦对所选出的单个真菌细胞进行激光显微切割，获得单个目的细胞，重复上述操作获取总数为1-100个的目的细胞；
(2)真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质；
1)将0.8M D-山梨醇溶液加入到装有上述目的细胞的离心管中，在4℃条件下浸泡目的细胞2小时；
2)配制复合预处理剂：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
3)配制混合酶处理剂：将蜗牛酶1-10mg/mL，溶壁酶1-10mg/mL，溶菌酶1-10mg/mL，纤维素酶1-10mg/mL四种酶中的至少两种酶按一定比例混合，当所述混合酶处理剂仅由两种酶混合组成时，其中至少包括蜗牛酶和溶壁酶中的一种；
4)将上述复合预处理剂加入装有目的细胞的离心管中，35℃处理细胞1小时；
5)上述离心管经离心后将细胞收集至管底，弃去处理剂；
6)向上述离心管中加入无菌水洗涤细胞两次，离心，弃去液体；
7)向上述离心管中加入混合酶处理剂，35-45℃处理3-12小时；
8)上述离心管经离心后将细胞收集至管底，弃去液体；
9)向上述离心管中加入PBS洗涤细胞一次，离心，弃去液体，保留4-10μl细胞悬液；
(3)微量真菌原生质gDNA的提取和扩增：利用单细胞全基因组扩增试剂盒对上述步骤中获得的4-10μl细胞悬液进行核酸提取及扩增反应，获得上述真菌gDNA；
(4)微量gDNA建库：利用DNA建库试剂盒对上述真菌gDNA进行建库；
(5)基因组测序：对上述建库的真菌gDNA进行全基因组测序；
(6)生物信息学分析：利用相关软件对测序获得的数据进行组装，得到组装结果与相关公开数据库进行比对，判定所检测真菌的种类。
进一步地，本发明方法可检测的真菌包括念珠菌属中的白色念珠菌、光滑念珠菌、克柔念珠菌、热带念珠菌、近平滑念珠菌、都柏林念珠菌；曲霉属中的烟曲霉、黄曲霉、土曲霉、构巢曲霉、黑曲霉、焦曲霉；隐球菌属中的新生隐球菌、格特隐球菌以及上述各菌种的变种；上述各菌种及其变种均属于临床医学中的致病菌，虽然是可被体外培养的，但由于患者用药等因素，在真实临床样本中难以被分离培养成功。
进一步地，本发明方法可检测的真菌还包括马拉色菌，该菌种属于苛养的真菌，对培养条件要求苛刻，需要在培养基中补充特殊营养成分才能够培养成功。
进一步地，本发明方法可检测的真菌还包括组织胞浆菌、伊蒙菌、申克氏孢子丝菌、马 尔尼菲青霉、巴西副球孢子菌、皮炎芽生菌、粗球孢子菌；上述菌种在体外难以培养成功。
作为一种优选方案，本发明方法中的第(2)步骤真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质，其中配制的混合酶处理剂为蜗牛酶6mg/mL和溶壁酶4mg/mL；向离心管中加入混合酶处理剂后，37℃处理6小时；最终保留4μl细胞悬液。
作为另一种优选方案，本发明方法中的第(2)步骤真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质，其中配制的混合酶处理剂为蜗牛酶6mg/mL和溶菌酶4mg/mL；向离心管中加入混合酶处理剂后，45℃处理10小时；最终保留5μl细胞悬液。
作为另一种优选方案，本发明方法中的第(2)步骤真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质，其中配制的混合酶处理剂为蜗牛酶4mg/mL、溶壁酶4mg/mL和溶菌酶4mg/mL；向离心管中加入混合酶处理剂后，35℃处理8小时；最终保留6μl细胞悬液。
作为另一种优选方案，本发明方法中的第(2)步骤真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质，其中配制的混合酶处理剂为蜗牛酶4mg/mL、溶壁酶3mg/mL和纤维素酶3mg/mL；向离心管中加入混合酶处理剂后，37℃处理3小时；最终保留6μl细胞悬液。
作为另一种优选方案，本发明方法中的第(2)步骤真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质，其中配制的混合酶处理剂为蜗牛酶4mg/mL、溶菌酶3mg/mL和纤维素酶3mg/mL；向离心管中加入混合酶处理剂后，37℃处理12小时；最终保留8μl细胞悬液。
作为另一种优选方案，本发明方法中的第(2)步骤真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质，其中配制的混合酶处理剂为溶壁酶5mg/mL、溶菌酶4mg/mL和纤维素酶3mg/mL；向离心管中加入混合酶处理剂后，37℃处理6小时；最终保留10μl细胞悬液。
作为另一种优选方案，本发明方法中的第(2)步骤真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质，其中配制的混合酶处理剂为蜗牛酶4mg/mL、溶壁酶2mg/mL、溶菌酶3mg/mL和纤维素酶4mg/mL；向离心管中加入混合酶处理剂后，37℃处理6小时；最终保留6μl细胞悬液。
此外，本发明还提供了一种根据本发明微量真菌检测方法制成的真菌检测试剂盒，该真菌检测试剂盒可适用于以下领域：工业生产、环境监测、空气检测、土壤检测、水质检测、食品检测、药品检测、化妆品检测、保健品检测以及医学检测。
有益效果
本发明的微量真菌检测方法及相应试剂盒克服了现有技术存在的许多不足，为从复杂样本中分离获取难以培养的真菌提供了方案；为微量真菌的高效破壁提供了优化的技术方案； 并为针对复杂样本中微量真菌进行基因组测序分析提供了整套的技术方案，最终实现了对微量真菌的高效检测。本发明方法可以直接应用于微量、疑难真菌样本或混合样本的分离、检测、鉴别及遗传信息的深入研究。根据本发明方法制成的真菌检测试剂盒可广泛应用于工业生产、环境监测、空气检测、土壤检测、水质检测、食品检测、药品检测、化妆品检测、保健品检测以及医学检测等多种领域。本发明技术的推广应用将会具有良好的市场前景并产生可观的经济及社会效益。
附图说明
图1为DNA质控检测电泳图，其中：“M”为DNA marker，1-3号为可建库的gDNA阳性对照，4-6号为本实验提取的真菌单细胞gDNA，样本和阳性对照的DNA都有≥10kb明显条带。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例1
1.微量格特隐球菌细胞的获取：显微涂片、激光切割，获得单个目的细胞
将微量样本涂于激光显微切割覆膜载玻片(Leica Membrane Slides PEN2.0微米或类似产 品)，用激光显微切割系统(Leica LMD7000或类似产品)在63倍放大下找到目的细胞，用激光强度40微焦(可设范围laser power 40±3)对所选出的单个真菌细胞进行激光显微切割，落于无菌的离心小管中，重复上述操作共获取10个真菌细胞(可获取细胞数范围1-100个)。
2.格特隐球菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质
1)将200μl 0.8M D-山梨醇溶液加入到上述离心管中，在4℃条件下浸泡细胞2小时；
2)配制复合预处理剂100ml：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
3)配制混合酶处理剂1ml：蜗牛酶6mg/mL，溶壁酶4mg/mL；
4)加入200μl复合预处理剂于离心管中，35℃处理细胞1小时；
5)5000rpm离心5min将细胞收集至管底，弃去处理剂；
6)加入200μl无菌水洗涤细胞两次，5000rpm离心5min，弃去液体；
7)加入200μl混合酶处理剂，37℃处理6小时；
8)5000rpm离心5min将细胞收集至管底，弃去液体；
9)加入50μl PBS洗涤细胞一次，5000rpm离心5min，弃去液体，保留4μl细胞悬液。
3.微量格特隐球菌原生质gDNA的提取和扩增
衔接步骤2中4μl细胞悬液，加入REPLI-g Single Cell Kit(德国Qiagen公司)中3μl Buffer D2，混匀并瞬时离心，65℃孵育10min；加入3μl试剂盒中Stop Solution，混匀并瞬时离心，暂时保存于冰上；每个扩增反应中加40μl PCR反应液(按试剂盒说明书配制：H2O sc 9μl，REPLI-g sc Reaction Buffer 29μl，REPLI-g sc DNA Polymerase 2μl)，30℃反应8小时；于65℃3min下使REPLI-g DNA聚合酶失活，即为提取的格特隐球菌gDNA。
上述10个格特隐球菌细胞的gDNA提取扩增后，经DNA质控检测达到二代测序建库要求，OD260/OD280：1.82，DNA浓度：75ng/μl，完整性>10kb，如附图1所示，其中“M”为DNA marker，1-3号为可建库的gDNA阳性对照，4-6号为本实验提取的真菌单细胞gDNA，样本和阳性对照的DNA都有≥10kb明显条带。以上结果说明单细胞的DNA提取扩增实验是成功的，符合二代测序建库要求，可以进行全基因组测序。
4.微量gDNA建库
按照试剂盒
Figure PCTCN2016093734-appb-000001
UltraDNA Library Prep Kit for
Figure PCTCN2016093734-appb-000002
(美国NEB公司)对以上格特隐球菌gDNA进行建库。
5.新一代基因组测序
用Illumina MiSeq仪器及相应试剂(MiSeq 600cycles Reagent V3美国Illumina公司)对以上建库的gDNA进行全基因组测序。
6.生物信息学分析
使用SPAdes软件对测序数据进行组装，得到组装结果与NCBI公开数据库nt库进行比对，判定是格特隐球菌。
实施例2
1.微量都柏林念珠菌细胞的获取：显微涂片、激光切割，获得单个目的细胞
将微量样本涂于激光显微切割覆膜载玻片(Leica Membrane Slides PEN2.0微米或类似产品)，用激光显微切割系统(Leica LMD7000或类似产品)在63倍放大下找到目的细胞，用激光强度43微焦(可设范围laser power 40±3)对所选出的单个真菌细胞进行激光显微切割，落于无菌的离心小管中，共获取1个真菌细胞(可获取细胞数范围1-100个)。
2.都柏林念珠菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质
1)将200μl 0.8M D-山梨醇溶液加入到上述离心管中，在4℃条件下浸泡细胞2小时；
2)配制复合预处理剂100ml：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
3)配制混合酶处理剂1ml：蜗牛酶6mg/mL，溶菌酶4mg/mL；
4)加入200μl复合预处理剂于离心管中，35℃处理细胞1小时；
5)5000rpm离心5min将细胞收集至管底，弃去处理剂；
6)加入200μl无菌水洗涤细胞两次，5000rpm离心5min，弃去液体；
7)加入200μl混合酶处理剂，45℃处理10小时；
8)5000rpm离心5min将细胞收集至管底，弃去液体；
9)加入50μl PBS洗涤细胞一次，5000rpm离心5min，弃去液体，保留5μl细胞悬液。
3.微量都柏林念珠菌原生质gDNA的提取和扩增
衔接步骤2中5μl细胞悬液，加入REPLI-g Single Cell Kit(德国Qiagen公司)中3μl Buffer D2，混匀并瞬时离心，65℃孵育10min；加入3μl试剂盒中Stop Solution，混匀并瞬时离心，暂时保存于冰上；每个扩增反应中加40μl PCR反应液(按试剂盒说明书配制：H2O sc 9μl，REPLI-g sc Reaction Buffer 29μl，REPLI-g sc DNA Polymerase 2μl)，30℃反应8小时；于65℃3min下使REPLI-g DNA聚合酶失活，即为提取的都柏林念珠菌gDNA。
4.微量gDNA建库
按照试剂盒
Figure PCTCN2016093734-appb-000003
UltraDNA Library Prep Kit for
Figure PCTCN2016093734-appb-000004
(美国NEB公司)对以上都柏林念珠菌gDNA进行建库。
5.新一代基因组测序
用Illumina MiSeq仪器及相应试剂(MiSeq 600cycles Reagent V3美国Illumina公司)对以上建库的gDNA进行全基因组测序。
6.生物信息学分析
使用SPAdes软件对测序数据进行组装，得到组装结果与NCBI公开数据库nt库进行比对，判定是都柏林念珠菌。
实施例3
1.微量马拉色菌细胞的获取：显微涂片、激光切割，获得单个目的细胞
将微量样本涂于激光显微切割覆膜载玻片(Leica Membrane Slides PEN2.0微米或类似产品)，用激光显微切割系统(Leica LMD7000或类似产品)在63倍放大下找到目的细胞，用激光强度38微焦(可设范围laser power 40±3)对所选出的单个真菌细胞进行激光显微切割，落于无菌的离心小管中，重复上述操作共获取60个真菌细胞(可获取细胞数范围1-100个)。
2.马拉色菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质
1)将200μl 0.8M D-山梨醇溶液加入到上述离心管中，在4℃条件下浸泡细胞2小时；
2)配制复合预处理剂100ml：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
3)配制混合酶处理剂1ml：蜗牛酶4mg/mL，溶壁酶4mg/mL，溶菌酶4mg/mL；
4)加入200μl复合预处理剂于离心管中，35℃处理细胞1小时；
5)5000rpm离心5min将细胞收集至管底，弃去处理剂；
6)加入200μl无菌水洗涤细胞两次，5000rpm离心5min，弃去液体；
7)加入200μl混合酶处理剂，35℃处理8小时；
8)5000rpm离心5min将细胞收集至管底，弃去液体；
9)加入50μl PBS洗涤细胞一次，5000rpm离心5min，弃去液体，保留6μl细胞悬液。
3.微量马拉色菌原生质gDNA的提取和扩增
衔接步骤2中6μl细胞悬液，加入REPLI-g Single Cell Kit(德国Qiagen公司)中3μl Buffer D2，混匀并瞬时离心，65℃孵育10min；加入3μl试剂盒中Stop Solution，混匀并瞬时离心，暂时保存于冰上；每个扩增反应中加40μl PCR反应液(按试剂盒说明书配制：H2O sc 9μl，REPLI-g sc Reaction Buffer 29μl，REPLI-g sc DNA Polymerase 2μl)，30℃反应8小时；于65℃3min下使REPLI-g DNA聚合酶失活，即为提取的马拉色菌gDNA。
4.微量gDNA建库
按照试剂盒
Figure PCTCN2016093734-appb-000005
UltraDNA Library Prep Kit for
Figure PCTCN2016093734-appb-000006
(美国NEB公司)对以 上马拉色菌gDNA进行建库。
5.新一代基因组测序
用Illumina MiSeq仪器及相应试剂(MiSeq 600cycles Reagent V3美国Illumina公司)对以上建库的gDNA进行全基因组测序。
6.生物信息学分析
使用SPAdes软件对测序数据进行组装，得到组装结果与NCBI公开数据库nt库进行比对，判定是马拉色菌。
实施例4
1.微量伊蒙菌细胞的获取：显微涂片、激光切割，获得单个目的细胞
将微量样本涂于激光显微切割覆膜载玻片(Leica Membrane Slides PEN2.0微米或类似产品)，用激光显微切割系统(Leica LMD7000或类似产品)在63倍放大下找到目的细胞，用激光强度40微焦(可设范围laser power 40±3)对所选出的单个真菌细胞进行激光显微切割，落于无菌的离心小管中，重复上述操作共获取20个真菌细胞(可获取细胞数范围1-100个)。
2.伊蒙菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质
1)将200μl 0.8M D-山梨醇溶液加入到上述离心管中，在4℃条件下浸泡细胞2小时；
2)配制复合预处理剂100ml：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
3)配制混合酶处理剂1ml：蜗牛酶4mg/mL，溶壁酶3mg/mL，纤维素酶3mg/mL；
4)加入200μl复合预处理剂于离心管中，35℃处理细胞1小时；
5)5000rpm离心5min将细胞收集至管底，弃去处理剂；
6)加入200μl无菌水洗涤细胞两次，5000rpm离心5min，弃去液体；
7)加入200μl混合酶处理剂，37℃处理3小时；
8)5000rpm离心5min将细胞收集至管底，弃去液体；
9)加入50μl PBS洗涤细胞一次，5000rpm离心5min，弃去液体，保留6μl细胞悬液。
3.微量伊蒙菌原生质gDNA的提取和扩增
衔接步骤2中6μl细胞悬液，加入REPLI-g Single Cell Kit(德国Qiagen公司)中3μl Buffer D2，混匀并瞬时离心，65℃孵育10min；加入3μl试剂盒中Stop Solution，混匀并瞬时离心，暂时保存于冰上；每个扩增反应中加40μl PCR反应液(按试剂盒说明书配制：H2O sc 9μl，REPLI-g sc Reaction Buffer 29μl，REPLI-g sc DNA Polymerase 2μl)，30℃反应8小时；于65℃3min下使REPLI-g DNA聚合酶失活，即为提取的伊蒙菌gDNA。
4.微量gDNA建库
按照试剂盒
Figure PCTCN2016093734-appb-000007
UltraDNA Library Prep Kit for
Figure PCTCN2016093734-appb-000008
(美国NEB公司)对以上伊蒙菌gDNA进行建库。
5.新一代基因组测序
用Illumina MiSeq仪器及相应试剂(MiSeq 600cycles Reagent V3美国Illumina公司)对以上建库的gDNA进行全基因组测序。
6.生物信息学分析
使用SPAdes软件对测序数据进行组装，得到组装结果与NCBI公开数据库nt库进行比对，判定是伊蒙菌。
实施例5
1.微量黑曲霉细胞的获取：显微涂片、激光切割，获得单个目的细胞
将微量样本涂于激光显微切割覆膜载玻片(Leica Membrane Slides PEN2.0微米或类似产品)，用激光显微切割系统(Leica LMD7000或类似产品)在63倍放大下找到目的细胞，用激光强度37微焦(可设范围laser power 40±3)对所选出的单个真菌细胞进行激光显微切割，落于无菌的离心小管中，重复上述操作共获取100个真菌细胞(可获取细胞数范围1-100个)。
2.黑曲霉细胞壁破壁：化学法与混合酶法结合提取真菌原生质
1)将200μl 0.8M D-山梨醇溶液加入到上述离心管中，在4℃条件下浸泡细胞2小时；
2)配制复合预处理剂100ml：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
3)配制混合酶处理剂1ml：蜗牛酶4mg/mL，溶菌酶3mg/mL，纤维素酶3mg/mL；
4)加入200μl复合预处理剂于离心管中，35℃处理细胞1小时；
5)5000rpm离心5min将细胞收集至管底，弃去处理剂；
6)加入200μl无菌水洗涤细胞两次，5000rpm离心5min，弃去液体；
7)加入200μl混合酶处理剂，37℃处理12小时；
8)5000rpm离心5min将细胞收集至管底，弃去液体；
9)加入50μl PBS洗涤细胞一次，5000rpm离心5min，弃去液体，保留8μl细胞悬液。
3.微量黑曲霉原生质gDNA的提取和扩增
衔接步骤2中8μl细胞悬液，加入REPLI-g Single Cell Kit(德国Qiagen公司)中3μl Buffer D2，混匀并瞬时离心，65℃孵育10min；加入3μl试剂盒中Stop Solution，混匀并瞬时离心，暂时保存于冰上；每个扩增反应中加40μl PCR反应液(按试剂盒说明书配制：H2O sc 9μl， REPLI-g sc Reaction Buffer 29μl，REPLI-g sc DNA Polymerase 2μl)，30℃反应8小时；于65℃3min下使REPLI-g DNA聚合酶失活，即为提取的黑曲霉gDNA。
4.微量gDNA建库
按照试剂盒
Figure PCTCN2016093734-appb-000009
UltraDNA Library Prep Kit for
Figure PCTCN2016093734-appb-000010
(美国NEB公司)对以上黑曲霉gDNA进行建库。
5.新一代基因组测序
用Illumina MiSeq仪器及相应试剂(MiSeq 600cycles Reagent V3美国Illumina公司)对以上建库的gDNA进行全基因组测序。
6.生物信息学分析
使用SPAdes软件对测序数据进行组装，得到组装结果与NCBI公开数据库nt库进行比对，判定是黑曲霉。
实施例6
1.微量皮炎芽生菌细胞的获取：显微涂片、激光切割，获得单个目的细胞
将微量样本涂于激光显微切割覆膜载玻片(Leica Membrane Slides PEN2.0微米或类似产品)，用激光显微切割系统(Leica LMD7000或类似产品)在63倍放大下找到目的细胞，用激光强度42微焦(可设范围laser power 40±3)对所选出的单个真菌细胞进行激光显微切割，落于无菌的离心小管中，重复上述操作共获取30个真菌细胞(可获取细胞数范围1-100个)。
2.皮炎芽生菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质
1)将200μl 0.8M D-山梨醇溶液加入到上述离心管中，在4℃条件下浸泡细胞2小时；
2)配制复合预处理剂100ml：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
3)配制混合酶处理剂1ml：溶壁酶5mg/mL，溶菌酶4mg/mL，纤维素酶3mg/mL；
4)加入200μl复合预处理剂于离心管中，35℃处理细胞1小时；
5)5000rpm离心5min将细胞收集至管底，弃去处理剂；
6)加入200μl无菌水洗涤细胞两次，5000rpm离心5min，弃去液体；
7)加入200μl混合酶处理剂，37℃处理6小时；
8)5000rpm离心5min将细胞收集至管底，弃去液体；
9)加入50μl PBS洗涤细胞一次，5000rpm离心5min，弃去液体，保留10μl细胞悬液。
3.微量皮炎芽生菌原生质gDNA的提取和扩增
衔接步骤2中10μl细胞悬液，加入REPLI-g Single Cell Kit(德国Qiagen公司)中3μl  Buffer D2，混匀并瞬时离心，65℃孵育10min；加入3μl试剂盒中Stop Solution，混匀并瞬时离心，暂时保存于冰上；每个扩增反应中加40μl PCR反应液(按试剂盒说明书配制：H2O sc9μl，REPLI-g sc Reaction Buffer 29μl，REPLI-g sc DNA Polymerase 2μl)，30℃反应8小时；于65℃3min下使REPLI-g DNA聚合酶失活，即为提取的皮炎芽生菌gDNA。
4.微量gDNA建库
按照试剂盒
Figure PCTCN2016093734-appb-000011
UltraDNA Library Prep Kit for
Figure PCTCN2016093734-appb-000012
(美国NEB公司)对以上皮炎芽生菌gDNA进行建库。
5.新一代基因组测序
用Illumina MiSeq仪器及相应试剂(MiSeq 600cycles Reagent V3美国Illumina公司)对以上建库的gDNA进行全基因组测序。
6.生物信息学分析
使用SPAdes软件对测序数据进行组装，得到组装结果与NCBI公开数据库nt库进行比对，判定是皮炎芽生菌。
实施例7
1.微量粗球孢子菌细胞的获取：显微涂片、激光切割，获得单个目的细胞
将微量样本涂于激光显微切割覆膜载玻片(Leica Membrane Slides PEN2.0微米或类似产品)，用激光显微切割系统(Leica LMD7000或类似产品)在63倍放大下找到目的细胞，用激光强度40微焦(可设范围laser power 40±3)对所选出的单个真菌细胞进行激光显微切割，落于无菌的离心小管中，重复上述操作共获取5个真菌细胞(可获取细胞数范围1-100个)。
2.粗球孢子菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质
1)将200μl 0.8M D-山梨醇溶液加入到上述离心管中，在4℃条件下浸泡细胞2小时；
2)配制复合预处理剂100ml：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
3)配制混合酶处理剂1ml：蜗牛酶4mg/mL，溶壁酶2mg/mL，溶菌酶3mg/mL，纤维素酶4mg/mL；
4)加入200μl复合预处理剂于离心管中，35℃处理细胞1小时；
5)5000rpm离心5min将细胞收集至管底，弃去处理剂；
6)加入200μl无菌水洗涤细胞两次，5000rpm离心5min，弃去液体；
7)加入200μl混合酶处理剂，37℃处理6小时；
8)5000rpm离心5min将细胞收集至管底，弃去液体；
9)加入50μl PBS洗涤细胞一次，5000rpm离心5min，弃去液体，保留6μl细胞悬液。
3.微量粗球孢子菌原生质gDNA的提取和扩增
衔接步骤2中6μl细胞悬液，加入REPLI-g Single Cell Kit(德国Qiagen公司)中3μl Buffer D2，混匀并瞬时离心，65℃孵育10min；加入3μl试剂盒中Stop Solution，混匀并瞬时离心，暂时保存于冰上；每个扩增反应中加40μl PCR反应液(按试剂盒说明书配制：H2O sc 9μl，REPLI-g sc Reaction Buffer 29μl，REPLI-g sc DNA Polymerase 2μl)，30℃反应8小时；于65℃3min下使REPLI-g DNA聚合酶失活，即为提取的粗球孢子菌gDNA。
4.微量gDNA建库
按照试剂盒
Figure PCTCN2016093734-appb-000013
UltraDNA Library Prep Kit for
Figure PCTCN2016093734-appb-000014
(美国NEB公司)对以上粗球孢子菌gDNA进行建库。
5.新一代基因组测序
用Illumina MiSeq仪器及相应试剂(MiSeq 600cycles Reagent V3美国Illumina公司)对以上建库的gDNA进行全基因组测序。
6.生物信息学分析
使用SPAdes软件对测序数据进行组装，得到组装结果与NCBI公开数据库nt库进行比对，判定是粗球孢子菌。

Claims (12)

  1. 一种利用单细胞测序检测微量真菌的方法，包括以下步骤：
    (1)微量真菌细胞的获取：将微量真菌样本涂于激光显微切割覆膜载玻片上，用激光显微切割系统找到目的细胞，用激光强度37-43微焦对所选出的单个真菌细胞进行激光显微切割，获得单个目的细胞，重复上述操作获取总数为1-100个的目的细胞；
    (2)真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质；
    1)将0.8M D-山梨醇溶液加入到装有上述目的细胞的离心管中，在4℃条件下浸泡目的细胞2小时；
    2)配制复合预处理剂：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
    3)配制混合酶处理剂：将蜗牛酶1-10mg/mL，溶壁酶1-10mg/mL，溶菌酶1-10mg/mL，纤维素酶1-10mg/mL四种酶中的至少两种酶按一定比例混合，当所述混合酶处理剂仅由两种酶混合组成时，其中至少包括蜗牛酶和溶壁酶中的一种；
    4)将上述复合预处理剂加入装有目的细胞的离心管中，35℃处理细胞1小时；
    5)上述离心管经离心后将细胞收集至管底，弃去处理剂；
    6)向上述离心管中加入无菌水洗涤细胞两次，离心，弃去液体；
    7)向上述离心管中加入混合酶处理剂，35-45℃处理3-12小时；
    8)上述离心管经离心后将细胞收集至管底，弃去液体；
    9)向上述离心管中加入PBS洗涤细胞一次，离心，弃去液体，保留4-10μl细胞悬液；
    (3)微量真菌原生质gDNA的提取和扩增：利用单细胞全基因组扩增试剂盒对上述步骤中获得的4-10μl细胞悬液进行核酸提取及扩增反应，获得上述真菌gDNA；
    (4)微量gDNA建库：利用DNA建库试剂盒对上述真菌gDNA进行建库；
    (5)基因组测序：对上述建库的真菌gDNA进行全基因组测序；
    (6)生物信息学分析：利用相关软件对测序获得的数据进行组装，得到组装结果与相关公开数据库进行比对，判定所检测真菌的种类。
  2. 如权利要求1所述利用单细胞测序检测微量真菌的方法，其特征在于：所述真菌包括念珠菌属中的白色念珠菌、光滑念珠菌、克柔念珠菌、热带念珠菌、近平滑念珠菌、都柏林念珠菌；曲霉属中的烟曲霉、黄曲霉、土曲霉、构巢曲霉、黑曲霉、焦曲霉；隐球菌属中的新生隐球菌、格特隐球菌以及上述各菌种的变种。
  3. 如权利要求1所述利用单细胞测序检测微量真菌的方法，其特征在于：所述真菌包括马拉色菌。
  4. 如权利要求1所述利用单细胞测序检测微量真菌的方法，其特征在于：所述真菌包括 组织胞浆菌、伊蒙菌、申克氏孢子丝菌、马尔尼菲青霉、巴西副球孢子菌、皮炎芽生菌、粗球孢子菌。
  5. 如权利要求1-4中任一项所述的利用单细胞测序检测微量真菌的方法，其特征在于：其中的第(2)步骤如下：
    (2)真菌细胞壁破壁：化学法与混合酶法结合提取真菌原生质；
    1)将0.8M D-山梨醇溶液加入到装有上述目的细胞的离心管中，在4℃条件下浸泡目的细胞2小时；
    2)配制复合预处理剂：将50mM Tris，5mM EDTA，5％β-巯基乙醇混合均匀；
    3)配制混合酶处理剂：蜗牛酶6mg/mL，溶壁酶4mg/mL；
    4)将上述复合预处理剂加入装有目的细胞的离心管中，35℃处理细胞1小时；
    5)上述离心管经离心后将细胞收集至管底，弃去处理剂；
    6)向上述离心管中加入无菌水洗涤细胞两次，离心，弃去液体；
    7)向上述离心管中加入混合酶处理剂，37℃处理6小时；
    8)上述离心管经离心后将细胞收集至管底，弃去液体；
    9)向上述离心管中加入PBS洗涤细胞一次，离心，弃去液体，保留4μl细胞悬液。
  6. 如权利要求1-4中任一项所述的利用单细胞测序检测微量真菌的方法，其特征在于：其中的第(2)步骤优选如下：
    3)配制混合酶处理剂：蜗牛酶6mg/mL，溶菌酶4mg/mL；
    7)向上述离心管中加入混合酶处理剂，45℃处理10小时；
    9)向上述离心管中加入PBS洗涤细胞一次，离心，弃去液体，保留5μl细胞悬液。
  7. 如权利要求1-4中任一项所述的利用单细胞测序检测微量真菌的方法，其特征在于：其中的第(2)步骤优选如下：
    3)配制混合酶处理剂：蜗牛酶4mg/mL，溶壁酶4mg/mL，溶菌酶4mg/mL；
    7)向上述离心管中加入混合酶处理剂，35℃处理8小时；
    9)向上述离心管中加入PBS洗涤细胞一次，离心，弃去液体，保留6μl细胞悬液。
  8. 如权利要求1-4中任一项所述的利用单细胞测序检测微量真菌的方法，其特征在于：其中的第(2)步骤优选如下：
    3)配制混合酶处理剂：蜗牛酶4mg/mL，溶壁酶3mg/mL，纤维素酶3mg/mL；
    7)向上述离心管中加入混合酶处理剂，37℃处理3小时；
    9)向上述离心管中加入PBS洗涤细胞一次，离心，弃去液体，保留6μl细胞悬液。
  9. 如权利要求1-4中任一项所述的利用单细胞测序检测微量真菌的方法，其特征在于：其中的第(2)步骤优选如下：
    3)配制混合酶处理剂：蜗牛酶4mg/mL，溶菌酶3mg/mL，纤维素酶3mg/mL；
    7)向上述离心管中加入混合酶处理剂，37℃处理12小时；
    9)向上述离心管中加入PBS洗涤细胞一次，离心，弃去液体，保留8μl细胞悬液。
  10. 如权利要求1-4中任一项所述的利用单细胞测序检测微量真菌的方法，其特征在于：其中的第(2)步骤优选如下：
    3)配制混合酶处理剂：溶壁酶5mg/mL，溶菌酶4mg/mL，纤维素酶3mg/mL；
    7)向上述离心管中加入混合酶处理剂，37℃处理6小时；
    9)向上述离心管中加入PBS洗涤细胞一次，离心，弃去液体，保留10μl细胞悬液。
  11. 如权利要求1-4中任一项所述的利用单细胞测序检测微量真菌的方法，其特征在于：其中的第(2)步骤优选如下：
    3)配制混合酶处理剂：蜗牛酶4mg/mL，溶壁酶2mg/mL，溶菌酶3mg/mL，纤维素酶4mg/mL；
    7)向上述离心管中加入混合酶处理剂，37℃处理6小时；
    9)向上述离心管中加入PBS洗涤细胞一次，离心，弃去液体，保留6μl细胞悬液。
  12. 一种根据权利要求1所述利用单细胞测序检测微量真菌的方法制成的真菌检测试剂盒，所述真菌检测试剂盒适用于以下领域：工业生产、环境监测、空气检测、土壤检测、水质检测、食品检测、药品检测、化妆品检测、保健品检测以及医学检测。
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