# US 2014/0155284 A1 · System for identification of microorganism and detection of infectious disease

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- Number: US 2014/0155284 A1
- Document ID: US20140155284A1
- Status: Published application
- Kind: A1
- Application number: 14/064,140
- Filed: 2013-10-26
- Published: 2014-06-05
- Earliest claimed: 2011-04-28
- Inventors: Jørn Erland Koch; Magnus Stougaard; Birgitta Ruth Knudsen; Sissel Juul; Kam Leong; Yi-Ping Ho; Felicie F. Andersen
- Web page: https://patentgrep.com/patent/US20140155284A1

## CPC

- C12Q 1/6893: for protozoa
- 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

Methods for the identification of microorganisms or infectious disorders are disclosed, comprising obtaining a suitable sample from sources such as persons, animals, plants, food, water or soil. The methods also comprise providing tailored nucleic acid substrate(s) designed to react with a type 1 topoisomerase from one or more microorganism(s) or infectious agent(s), and incubating said substrate with said sample, or extracts or preparations from the sample, so that the substrate is processed by said topoisomerase if said microorganism(s) or infectious agent(s) is present in the sample. Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are also provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted by that enzyme.

## Claims

### Claim 1

A method of identifying a type I topoisomerase-expressing microorganism in a sample, said method comprising
i) providing the sample
ii) providing a nucleic acid substrate targeted by a type I topoisomerase of said microorganism,
iii) bringing the sample of step i) in contact with the nucleic acid substrate of step ii), and
iv) detecting the nucleic acid substrate processed by said type I topoisomerase of said microorganism,
wherein the presence of the processed nucleic acid substrate is indicative of said microorganism.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said microorganism is a pathogenic microorganism and/or a parasitic microorganism.

### Claim at position 3

3-5. (canceled)

### Claim 6 (depends on 2)

The method according to claim 2, wherein said microorganism is selected from the Plasmodium Genus.

### Claim 7 (depends on 6)

The method according to claim 6, wherein said microorganism is Plasmodium falciparum.

### Claim at position 6

8-11. (canceled)

### Claim 12 (depends on 1)

The method according to claim 1, wherein the quantitative presence of said microorganism in said sample is determined.

### Claim at position 8

13-14. (canceled)

### Claim 15 (depends on 1)

The method according to claim 1, wherein said sample is a human sample.

### Claim at position 10

16-17. (canceled)

### Claim 18 (depends on 1)

The method according to claim 1, wherein said sample is blood plasma, blood serum, sputum, saliva, urine, cell smear, faeces, cerebrospinal fluid, or a biopsy.

### Claim 19 (depends on 1)

The method according to claim 1, wherein said sample is depleted for divalent cations.

### Claim 20 (depends on 1)

The method according to claim 1, wherein said nucleic acid substrate is predominantly targeted by type I topoisomerase of said microorganism and to a lesser extent by any type I topoisomerase native to said sample.

### Claim 21

(canceled)

### Claim 22 (depends on 1)

The method according to claim 1, wherein said nucleic acid substrate is double stranded.

### Claim 23 (depends on 22)

The method according to claim 22, wherein said double stranded nucleic acid substrate is provided as a single nucleic acid, which folds into a secondary hairpin structure comprising a double-stranded target region.

### Claim 24

(canceled)

### Claim 25 (depends on 6)

The method according to claim 6, wherein said nucleic acid substrate comprises a sequence selected from any one of SEQ ID NO: 8-19, a sequence at least 90% identical thereto, or a part of at least 5 consecutive nucleotides of any of said sequences.

### Claim 26 (depends on 25)

The method according to claim 25, wherein said nucleic acid substrate comprises the sequence TCTAGTAAG-(N)X-CTTA or ATTTTTCTA-(N)X-TAGA, where N is A, T, C, or G, and x is between 5 and 500 (SEQ ID NOs: 18 or 19).

### Claim 27 (depends on 26)

The method according to claim 26, wherein said nucleic acid substrate comprises a sequence, with at least 90% identity to any one of SEQ ID NOs: 8-17, and which comprise the sequence TCTAGTAAG-(N)X-CTTA or ATTTTTCTA-(N)X-TAGA, where N is A, T, C, or G, and x is between 5 and 500 (SEQ ID NOs: 18 or 19).

### Claim at position 21

28-30. (canceled)

### Claim 31 (depends on 1)

The method according to claim 1, wherein said nucleic acid substrate is processed by cleavage and/or ligation by said type I topoisomerase.

### Claim 32 (depends on 31)

The method according to claim 31, wherein said ligation is intramolecular ligation of the 3′-terminus of the nucleic acid substrate to the 5′-terminus of the nucleic acid substrate, thereby generating a circular nucleic acid product.

### Claim 33

(canceled)

### Claim 34 (depends on 32)

The method according to claim 32, wherein said ligation is catalyzed by said type I topoisomerase of said microorganism, by a heterogeneous ligase and/or by a recombinant ligase.

### Claim 35 (depends on 1)

The method according to claim 1, wherein said processed nucleic acid substrate is detected by southern blotting, polymerase chain reaction, RT-PCR, qPCR, RFLD, primer extension, DNA array technology, a linear amplification technique, isothermal amplification and/or rolling circle amplification.

### Claim 36

A method of identifying a type I topoisomerase-expressing microorganism in a sample, said method comprising
i) providing the sample
ii) providing a nucleic acid substrate targeted by a type I topoisomerase of said microorganism,
iii) bringing the sample of step i) in contact with the nucleic acid substrate of step ii),
iv) detecting the nucleic acid substrate processed by said type I topoisomerase of said microorganism,
wherein the presence of the processed nucleic acid substrate is indicative of said microorganism and said processed nucleic acid substrate is detected by rolling circle amplification.

### Claim 37 (depends on 36)

The method of claim 36, wherein said nucleic acid rolling circle amplification is performed by
i) providing at least one oligonucleotide primer, which is capable of hybridizing to the circularized nucleic acid substrate,
ii) hybridizing the at least one oligonucleotide primer to the circularized nucleic acid substrate,
iii) providing a nucleic acid polymerase and nucleotides
iv) generating a rolling circle amplification product by extending the at least one oligonucleotide primer using the circularized nucleic acid substrate as template, and
v) detecting the rolling circle amplification product.

### Claim 38 (depends on 37)

The method according to claim 37, wherein said at least one oligonucleotide primer is selected from SEQ ID NO: 23-24.

### Claim 39 (depends on 37)

The method according to claim 37, wherein said nucleic acid substrate, oligonucleotide primer, nucleic acid polymerase and/or nucleotides is immobilized on a solid support.

### Claim 40 (depends on 39)

The method according to claim 39, wherein said solid support is a glass surface, a magnetic bead, a lateral flow test strip, or a dipstick.

### Claim 41 (depends on 37)

The method according to claim 37, wherein one or more of said nucleotides comprises one or more detectable labels.

### Claim 42 (depends on 41)

The method according to claim 41, wherein said rolling circle amplification product is detected via its incorporation of said nucleotides comprising one or more detectable labels.

### Claim 43 (depends on 37)

The method according to claim 37, wherein said rolling circle amplification product is detected by hybridization of a labelled nucleic acid probe to multiple sites of the rolling circle amplification product.

### Claim 44 (depends on 43)

The method according to claim 43, wherein said nucleic acid probe is labelled with one or more fluorescent dyes, radioactive nucleotides and/or biotinylated nucleotides.

### Claim 45 (depends on 43)

The method according to claim 43, wherein said nucleic acid probe is coupled to an enzyme, which is capable of converting a substrate into a detectable product.

### Claim 46 (depends on 45)

The method according to claim 45, wherein said enzyme is fused with streptavidin, and coupled to said nucleic acid probe via interaction with said biotinylated nucleotides incorporated in the nucleic acid probe.

### Claim 47 (depends on 45)

The method according to claim 45, wherein said enzyme is horse-radish peroxidase.

### Claim at position 39

48-145. (canceled)