PATENT CLAIM ANALYSIS

Application Number: 16313906
Application Type: Utility
Filing Date: 2018-12
Publication Date: 2019-10
Patent Classification: ["506", "009000"]

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.

Claim (Index 1):
A method for detecting trace fungi using a single-cell sequencing, comprising the following steps:\n (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 the 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;\n 1) adding 0.8 M D-sorbitol solution into a centrifuge tube containing the target cells to immerse the target cells at 4\u00b0 C. for 2 h; \n 2) preparation of a composite pretreatment agent: mixing 50 mM Tris, 5 mM EDTA and 5% \u03b2-mercaptoethanol evenly; \n 3) preparation of a mixed enzyme treatment agent: preparing the mixed enzyme treatment agent according to any one of the following formula:\n A: 6 mg/mL of snail enzyme and 4 mg/mL of lywallzyme, \n B: 6 mg/mL of snail enzyme and 4 mg/mL of lysozyme, \n C: 4 mg/mL of snail enzyme, 4 mg/mL of lywallzyme and 4 mg/mL of lysozyme, \n D: 4 mg/mL of snail enzyme, 3 mg/mL of lywallzyme and 3 mg/mL of cellulase, \n E: 4 mg/mL of snail enzyme, 3 mg/mL of lysozyme and 3 mg/mL of cellulase, \n F: 5 mg/mL of lywallzyme, 4 mg/mL of lysozyme and 3 mg/mL of cellulase, and \n G: 4 mg/mL of snail enzyme, 2 mg/mL of lywallzyme, 3 mg/mL of lysozyme and 4 mg/mL of cellulase; \n \n 4) adding the above mentioned composite pretreatment agent into the centrifuge tube containing the target cells to treat the target cells at 35\u00b0 C. for 1 h; \n 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; \n 6) adding sterile water into the centrifuge tube to wash the target cells twice, centrifuging, and discarding liquid; \n 7) adding the mixed enzyme treatment agent into the centrifuge tube to treat the target cells at 35-45\u00b0 C. for 3-12 h; \n 8) subjecting the centrifuge tube to a centrifugation to collect the target cells to a bottom of the centrifuge tube, then discarding liquid; and \n 9) adding PBS (phosphate-buffered saline) into the centrifuge tube to wash the target cells once, centrifuging, discarding liquid, and retaining 4-10 \u03bcl of cell suspension; \n (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 \u03bcl 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.

Metadata:
- Claim Count in Document: 1.0
- Percentile: 98.0
- Lexical Diversity: 2.01235
- Patent Class: 506.0
- Transitional Phrase Type: open
- Component Type: 1
- Foreign Priority: True
- Related Applications: ['14117035', '11212812', '14648161', '15612091', '13833872']

Analysis Scores:
- 35 USC 101 Eligibility (BERT): 0.4997814467923873
- 35 USC 102 Novelty (BERT): 0.5061079165115789
- Combined Prediction Score: 0.5004140937643065
- Mean Citation Score: 175.581828
- Max Citation Score: 194.80453
- Similarity Product: 118.23438881708384

Labels:
- Claim Label 101: 1
- Claim Label 102: 1
- Claim Label 103: 1
- Claim Label 112: 1
- Combined Label: 1
- Label 101 Adjusted: 1

Dataset: test