Method for detecting trace fungi using single-cell sequencing and kit thereof

• US 10,557,176 B2
• Filed: 08/05/2016
• Issued: 02/11/2020
• Est. Priority Date: 07/08/2016
• Status: Active Grant

• Current Assignee: Beijing Institute of Radiation Medicine
• Original Assignee: Beijing Institute of Radiation Medicine
• Inventors: Yang, Ying, Wang, Shengqi, Zhou, Zhe, Chen, Min, Wang, Peng, Li, Zhen, Li, Zongwei
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US16/313,906
• Publication Number:
  US 20190323092A1
• Time in Patent Office: 1,285 Days
• CPC Class Codes: C12N 1/145 Fungal isolates
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12R 2001/645 Fungi ; Processes using fungi

Abstract

A method for detecting trace fungi using single-cell sequencing and a fungi detection kit prepared by the method are disclosed. The method includes the steps of obtaining trace fungal cells, extracting fungal protoplasm by breaking the cell walls of fungi, extracting gDNA from trace fungal protoplasm and amplifying the gDNA, constructing trace gDNA library, genome sequencing, bioinformatics analysis and comparison, and determining the species of detected fungi, etc. The method realizes the high-efficiency detection of trace fungi, and can be directly applied to the isolation, detection, identification of trace and difficult-to-identify fungal samples or mixed samples, and in-depth study of genetic information. The fungi detection method and the kit are applicable to industrial production, environmental monitoring, air testing, soil testing, water quality testing, food testing, drug testing, cosmetics testing, health care products testing and medical testing, and other fields.

Claims

1. A method for detecting trace fungi using a single-cell sequencing, comprising the following steps:

(1) acquisition of trace fungal cells;

coating trace fungi samples onto a membrane glass slide for laser microdissection, finding target cells using a laser microdissection system, and performing the laser microdissection on one of the target cells by a laser with a laser power of 37-43 micro joules to obtain a single target cell;

wherein the above operation is repeated to obtain the target cells with a total number of 1-100;

(2) wall-breaking of fungal cell walls;

extraction of fungal protoplasm by a chemical method combined with a mixed enzymatic method;

1) adding 0.8 M D-sorbitol solution into a centrifuge tube containing the target cells to immerse the target cells at 4°

C. for 2 h;

2) preparation of a composite pretreatment agent;

mixing 50 mM Tris, 5 mM EDTA and 5% β

-mercaptoethanol evenly;

3) preparation of a mixed enzyme treatment agent;

preparing the mixed enzyme treatment agent according to any one of the following formula;

A;

6 mg/mL of snail enzyme and 4 mg/mL of lywallzyme,

B;

6 mg/mL of snail enzyme and 4 mg/mL of lysozyme,

C;

4 mg/mL of snail enzyme, 4 mg/mL of lywallzyme and 4 mg/mL of lysozyme,

D;

4 mg/mL of snail enzyme, 3 mg/mL of lywallzyme and 3 mg/mL of cellulase,

E;

4 mg/mL of snail enzyme, 3 mg/mL of lysozyme and 3 mg/mL of cellulase,

F;

5 mg/mL of lywallzyme, 4 mg/mL of lysozyme and 3 mg/mL of cellulase, and

G;

4 mg/mL of snail enzyme, 2 mg/mL of lywallzyme, 3 mg/mL of lysozyme and 4mg/mL of cellulase;

4) adding the composite pretreatment agent into the centrifuge tube containing the target cells to treat the target cells at 35°

C. for 1 h;

5) subjecting the centrifuge tube to a centrifugation to collect the target cells to a bottom of the centrifuge tube, then discarding the composite pretreatment agent;

6) adding sterile water into the centrifuge tube to wash the target cells twice, centrifuging, and discarding liquid;

7) adding the mixed enzyme treatment agent into the centrifuge tube to treat the target cells at 35-45°

C. for 3-12 h;

8) subjecting the centrifuge tube to a centrifugation to collect the target cells to a bottom of the centrifuge tube, then discarding liquid; and

9) adding PBS (phosphate-buffered saline) into the centrifuge tube to wash the target cells once, centrifuging, discarding liquid, and retaining 4-10 μ

l of cell suspension;

(3) extraction fungal gDNA from the fungal protoplasm and amplification of the fungal gDNA;

using a single-cell whole genome amplification kit to perform a nucleic acid extraction and amplification reaction on the 4-10 μ

l cell suspension obtained in the step

     9) to obtain the fungal gDNA;

(4) library construction of trace gDNA;

constructing a library of the fungal gDNA using a DNA library construction kit;

(5) genome sequencing;

performing whole genome sequencing on the fungal gDNA after constructing the library; and

(6) bioinformatics analysis;

assembling data obtained from sequencing by using a relevant software to obtain assembled results, and the assembled results are compared with a relevant public database to determine a species of the trace fungi.

2. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the trace fungi are selected from the group consisting of Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, and Candida dubliniensis, within the genus Candida;

Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, and Aspergillus ustus, within the genus Aspergillus;

Cryptococcus neoformans and Cryptococcus gattii, within the genus Cryptococcus.

3. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the trace fungi comprise Malassezia sp.

4. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the trace fungi are selected from the group consisting of Histoplasma sp., Emmonsia sp., Sporothrix schenckii, Penicillium marneffei, Paracoccidiodes brasiliensis, Blastomycosis dermatitidis, and Coccidioides immitis.

5. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the step (2) is as follows:

(2) wall-breaking of the fungi cell walls;

extraction of the fungal protoplasm by the chemical method combined with the mixed enzymatic method;

1) adding the 0.8 M D-sorbitol solution into the centrifuge tube containing the target cells to immerse the target cells at 4°

C. for 2 h;

2) preparation of the composite pretreatment agent;

mixing the 50 mM Tris, the 5 mM EDTA and the 5% (β

-mercaptoethanol evenly;

3) preparation of the mixed enzyme treatment agent;

mixing the 6 mg/mL of snail enzyme, and the 4 mg/mL of lywallzyme;

4) adding the composite pretreatment agent into the centrifuge tube to treat the target cells at 35°

C. for 1 h;

5) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the composite pretreatment agent;

6) adding the sterile water to wash the target cells twice, centrifuging, and discarding the liquid;

7) adding the mixed enzyme treatment agent to treat the target cells at 37°

C. for 6 h;

8) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the liquid; and

9) adding the PBS into the centrifuge tube to wash the target cells once, centrifuging, discarding the liquid, and retaining 4 μ

l of the cell suspension.

6. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the step (2) is as follows:

(2) wall-breaking of the fungi cell walls;

extraction of the fungal protoplasm by the chemical method combined with the mixed enzymatic method;

1) adding the 0.8 M D-sorbitol solution into the centrifuge tube containing the target cells to immerse the target cells at 4°

C. for 2 h;

2) preparation of the composite pretreatment agent;

mixing the 50 mM Tris, the 5 mM EDTA and the 5% (β

-mercaptoethanol evenly;

3) preparation of the mixed enzyme treatment agent;

mixing the 6 mg/mL of snail enzyme, and the 4 mg/mL of lywallzyme;

4) adding the composite pretreatment agent into the centrifuge tube to treat the target cells at 35°

C. for 1 h;

5) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the composite pretreatment agent;

6) adding the sterile water to wash the target cells twice, centrifuging, and discarding the liquid;

7) adding the mixed enzyme treatment agent into the centrifuge tube to treat the target cells at 45°

C. for 10 h;

8) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the liquid; and

9) adding the PBS into the centrifuge tube to wash the target cells once, centrifuging, discarding the liquid, and retaining 5 μ

l of the cell suspension.

7. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the step (2) is as follows:

(2) wall-breaking of the fungi cell walls;

extraction of the fungal protoplasm by the chemical method combined with the mixed enzymatic method;

1) adding the 0.8 M D-sorbitol solution into the centrifuge tube containing the target cells to immerse the target cells at 4°

C. for 2 h;

2) preparation of the composite pretreatment agent;

mixing the 50 mM Tris, the 5 mM EDTA and the 5% (β

-mercaptoethanol evenly;

3) preparation of the mixed enzyme treatment agent;

mixing the 4 mg/mL of snail enzyme, the 4 mg/mL of lywallzyme, and the 4 mg/mL of lysozyme;

4) adding the composite pretreatment agent into the centrifuge tube to treat the target cells at 35°

C. for 1 h;

5) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the composite pretreatment agent;

6) adding the sterile water to wash the target cells twice, centrifuging, and discarding the liquid;

7) adding the mixed enzyme treatment agent into the centrifuge tube to treat the target cells at 35°

C. for 8h;

8) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the liquid; and

9) adding the PBS into the above-mentioned centrifuge tube to wash the target cells once, centrifuging, discarding the liquid, and retaining 6 μ

l of the cell suspension.

8. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the step (2) is as follows:

(2) wall-breaking of the fungi cell walls;

extraction of the fungal protoplasm by the chemical method combined with the mixed enzymatic method;

1) adding the 0.8 M D-sorbitol solution into the centrifuge tube containing the target cells to immerse the target cells at 4°

C. for 2 h;

2) preparation of the composite pretreatment agent;

mixing the 50 mM Tris, the 5 mM EDTA and the 5% (β

-mercaptoethanol evenly;

3) preparation of the mixed enzyme treatment agent;

mixing the 4 mg/mL of snail enzyme, the 3 mg/mL of lywallzyme, and the 3 mg/mL of cellulase;

4) adding the composite pretreatment agent into the centrifuge tube to treat the target cells at 35°

C. for 1 h;

5) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the composite pretreatment agent;

6) adding the sterile water to wash the target cells twice, centrifuging, and discarding the liquid;

7) adding the mixed enzyme treatment agent into the centrifuge tube to treat the target cells at 37°

C. for 3 h;

8) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the liquid; and

9) adding the PBS into the centrifuge tube to wash the target cells once, centrifuging, discarding the liquid, and retaining 6 μ

l of the cell suspension.

9. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the step (2) is as follows:

(2) wall-breaking of the fungi cell walls;

extraction of the fungal protoplasm by the chemical method combined with the mixed enzymatic method;

1) adding the 0.8 M D-sorbitol solution into the centrifuge tube containing the target cells to immerse the target cells at 4°

C. for 2 h;

2) preparation of the composite pretreatment agent;

mixing the 50 mM Tris, the 5 mM EDTA and the 5% (β

-mercaptoethanol evenly;

3) preparation of the mixed enzyme treatment agent;

mixing the 4 mg/mL of snail enzyme, the 3 mg/mL of lysozyme, and the 3 mg/mL of cellulase;

4) adding the composite pretreatment agent into the centrifuge tube to treat the target cells at 35°

C. for 1 h;

5) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the composite pretreatment agent;

6) adding the sterile water to wash the target cells twice, centrifuging, and discarding the liquid;

7) adding the mixed enzyme treatment agent into the centrifuge tube to treat the target cells at 37°

C. for 12 h;

8) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the liquid; and

9) adding the PBS into the centrifuge tube to wash the target cells once, centrifuging, discarding the liquid, and retaining 8 μ

l of the cell suspension.

10. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the step (2) is as follows:

(2) wall-breaking of the fungi cell walls;

extraction of the fungal protoplasm by the chemical method combined with the mixed enzymatic method;

1) adding the 0.8 M D-sorbitol solution into the centrifuge tube containing the target cells to immerse the target cells at 4°

C. for 2 h;

2) preparation of the composite pretreatment agent;

mixing the 50 mM Tris, the 5 mM EDTA and the 5% (β

-mercaptoethanol evenly;

3) preparation of the mixed enzyme treatment agent;

mixing the 5 mg/mL lywallzyme, the 4 mg/mL of lysozyme, and the 3 mg/mL of cellulase;

4) adding the composite pretreatment agent into the centrifuge tube to treat the target cells at 35°

C. for 1 h;

5) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the composite pretreatment agent;

6) adding the sterile water to wash the target cells twice, centrifuging, and discarding the liquid;

7) adding the mixed enzyme treatment agent into the centrifuge tube to treat the target cells at 37°

C. for 6 h;

8) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the liquid; and

9) adding the PBS into the centrifuge tube to wash the target cells once, centrifuging, discarding the liquid, and retaining 10 μ

l of the cell suspension.

11. The method for detecting the trace fungi using the single-cell sequencing according to claim 1, wherein the step (2) is as follows:

(2) wall-breaking of the fungi cell walls;

extraction of the fungal protoplasm by the chemical method combined with the mixed enzymatic method;

1) adding the 0.8 M D-sorbitol solution into the centrifuge tube containing the target cells to immerse the target cells at 4°

C. for 2 h;

2) preparation of the composite pretreatment agent;

mixing the 50 mM Tris, the 5 mM EDTA and the 5% (β

-mercaptoethanol evenly;

3) preparation of the mixed enzyme treatment agent;

mixing the 4 mg/mL of snail enzyme, the 2 mg/mL of lywallzyme, the 3 mg/mL of lysozyme, and the 4 mg/mL of cellulase;

4) adding the composite pretreatment agent into the centrifuge tube to treat the target cells at 35°

C. for 1 h;

5) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the composite pretreatment agent;

6) adding the sterile water to wash the target cells twice, centrifuging, and discarding the liquid;

7) adding the mixed enzyme treatment agent into the centrifuge tube to treat the target cells at 37°

C. for 6 h;

8) subjecting the centrifuge tube to the centrifugation to collect the target cells to the bottom of the centrifuge tube, then discarding the liquid; and

9) adding the PBS into the centrifuge tube to wash the target cells once, centrifuging, discarding the liquid, and retaining 6 μ

l of the cell suspension.

12. The method for detecting the trace fungi using the single-cell sequencing according to claim 5, wherein the trace fungi are selected from the group consisting of Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, and Candida dubliniensis, within the genus Candida;

Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, and Aspergillus ustus, within the genus Aspergillus;

Cryptococcus neoformans and Cryptococcus gattii, within the genus Cryptococcus.

13. The method for detecting the trace fungi using the single-cell sequencing according to claim 5, wherein the trace fungi comprise Malassezia sp.

14. The method for detecting the trace fungi using the single-cell sequencing according to claim 5, wherein the trace fungi are selected from the group consisting of Histoplasma sp., Emmonsia sp., Sporothrix schenckii, Penicillium marneffei, Paracoccidiodes brasiliensis, Blastomycosis dermatitidis, and Coccidioides immitis.

15. The method for detecting the trace fungi using the single-cell sequencing according to claim 6, wherein the trace fungi are selected from the group consisting of Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, and Candida dubliniensis, within the genus Candida;

Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, and Aspergillus ustus, within the genus Aspergillus;

Cryptococcus neoformans and Cryptococcus gattii, within the genus Cryptococcus.

16. The method for detecting the trace fungi using the single-cell sequencing according to claim 6, wherein the trace fungi comprise Malassezia sp.

17. The method for detecting the trace fungi using the single-cell sequencing according to claim 6, wherein the trace fungi are selected from the group consisting of Histoplasma sp., Emmonsia sp., Sporothrix schenckii, Penicillium marneffei, Paracoccidiodes brasiliensis, Blastomycosis dermatitidis, and Coccidioides immitis.

18. The method for detecting the trace fungi using the single-cell sequencing according to claim 7, wherein the trace fungi are selected from the group consisting of Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, and Candida dubliniensis, within the genus Candida;

Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, and Aspergillus ustus, within the genus Aspergillus;

Cryptococcus neoformans and Cryptococcus gattii, within the genus Cryptococcus.

19. The method for detecting the trace fungi using the single-cell sequencing according to claim 7, wherein the trace fungi comprise Malassezia sp.

20. The method for detecting the trace fungi using the single-cell sequencing according to claim 7, wherein the trace fungi are selected from the group consisting of Histoplasma sp., Emmonsia sp., Sporothrix schenckii, Penicillium marneffei, Paracoccidiodes brasiliensis, Blastomycosis dermatitidis, and Coccidioides immitis.