# US 7,745,160 B2 · Method of amplifying ATP and use thereof

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- Number: US 7,745,160 B2
- Document ID: US7745160B2
- Status: Granted
- Kind: B2
- Application number: 10/566,957
- Filed: 2004-07-27
- Published: 2008-09-04
- Granted: 2010-06-29
- Earliest claimed: 2003-07-29
- Assignees: Japan Science and Technology Agency
- Inventors: Akio Kuroda
- Web page: https://patentgrep.com/patent/US7745160B2

## CPC

- C12N 9/1229: {Phosphotransferases with a phosphate group as acceptor (2.7.4)}
- C12N 15/62: DNA sequences coding for fusion proteins
- C12P 19/32: having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
- C12Q 1/485: {involving kinase}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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

The ATP amplification method of the present invention is a method for amplifying and detecting a very trace amount of exogenous ATP by allowing a fusion protein (PPK-ADK) of a polyphosphate kinase and an adenylate kinase, the fusion protein not containing ADP, to act on a mixture of ATP, AMP, and a polyphosphate compound. The present invention also provides an ultrasensitive ATP amplification method by which ATP at a single cell level can be amplified and detected, and an ultrasensitive microbial assay based on this ATP amplification method.

## Claims

### Claim 1

A method for amplifying adenosine triphosphate comprising: allowing a fusion protein that has a polyphosphate kinase and an adenylate kinase in this order from the N-terminal to act on a mixture containing adenosine triphosphate, adenosine monophosphate, and a polyphosphate compound thereby amplifying adenosine triphosphate in the mixture,
wherein the fusion protein has been subjected to an apyrase treatment and a pyrophosphate treatment so as to remove adenosine diphosphate bound to the fusion protein.

### Claim 2

A method for detecting adenosine triphosphate comprising:
allowing a fusion protein that has a polyphosphate kinase and an adenylate kinase in this order from the N-terminal to act on a mixture of adenosine triphosphate, adenosine monophosphate, and a polyphosphate compound thereby amplifying adenosine triphosphate in the mixture; and
detecting the amplified adenosine triphosphate,
wherein the fusion protein has been subjected to an apyrase treatment and a pyrophosphate treatment so as to remove adenosine diphosphate bound to the fusion protein.

### Claim 3

A method for detecting the presence of a microorganism comprising:
treating a sample containing a microorganism to prepare a sample containing adenosine triphosphate;
adding the sample containing adenosine triphosphate to an adenosine triphosphate amplification system thereby amplifying adenosine triphosphate in the sample;
detecting the amplified adenosine triphosphate; and
detecting the presence of the microorganism by the presence of the detected adenosine triphosphate,
wherein the adenosine triphosphate amplification system comprises adenosine monophosphate, a polyphosphate compound, and a fusion protein that has a polyphosphate kinase and an adenylate kinase in this order from the N-terminal,
wherein the fusion protein has been subjected to an apyrase treatment and a pyrophosphate treatment so as to remove adenosine diphosphate bound to the fusion protein.