# US 10,370,729 B2 · Method for detecting fungi, reaction solution for PCR, and carrier for detecting fungi

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- Number: US 10,370,729 B2
- Document ID: US10370729B2
- Status: Granted
- Kind: B2
- Application number: 14/098,260
- Filed: 2013-12-05
- Published: 2014-05-22
- Granted: 2019-08-06
- Earliest claimed: 2011-06-09
- Assignees: Toyo Seikan Group Holdings, Ltd.
- Applicants: Toyo Seikan Group Holdings, Ltd.
- Inventors: Atsunori Isshiki; Suguru Tanabe; Hitoshi Takeharu; Mami Kokaji
- Web page: https://patentgrep.com/patent/US10370729B2

## CPC

- C12Q 1/6895: for plants, fungi or algae
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method for detecting fungi includes amplifying DNA fragments containing target regions in fungal DNA to confirm the presence or absence of an amplified product. As the target regions, both of the ITS region and the β-tubulin gene are used, and by using a primer set for amplifying the β-tubulin gene and a primer set for amplifying the ITS region in a reaction solution for PCR for amplifying the target regions, both of the target regions are simultaneously amplified according to one or two or more types of fungi.

## Claims

### Claim 1

A method for detecting fungi comprising:
preparing a PCR reaction solution comprising genomic DNA from collected fungi, a primer set for amplifying target regions of β-tubulin genes, and a primer set for amplifying target regions of ITS regions;
wherein, in the PCR reaction solution, the concentration ratio of the primer set for amplifying the target regions of the β-tubulin genes to the primer set for amplifying the target regions of the ITS regions is 1:0.5 to 1:0.25,
amplifying DNA fragments from the collected fungi by a multiplex PCR in the PCR reaction solution that includes a label,
hybridizing the labeled amplification products from the multiplex PCR to a DNA chip in which a plurality of probes selected from DNA of fungi to be detected are fixed; and
detecting the presence of fungi by measuring the combined label intensities of the labeled amplification product of the ITS region and the labeled amplification product of the β-tubulin gene hybridized on the DNA chip.

### Claim 2 (depends on 1)

The method for detecting fungi according to claim 1, wherein the label is a fluorescent label.

### Claim 3 (depends on 1)

The method for detecting fungi according to claim 1, wherein, in the PCR reaction solution, the primer set for amplifying the target regions of the ITS regions is a primer set comprising a forward primer comprising a base sequence represented by SEQ ID NO:1 and a reverse primer comprising a base sequence represented by SEQ ID NO:2, and the primer set for amplifying the target regions of the β-tubulin genes is a primer set comprising a forward primer comprising a base sequence represented by SEQ ID NO:3 and a reverse primer comprising a base sequence represented by SEQ ID NO:4.

### Claim 4 (depends on 3)

The method for detecting fungi according to claim 3, wherein, in the PCR reaction solution, a primer comprising a base sequence represented by sequence No. 5 is further used as a forward primer.