# US 2020/0399716 A1 · Compositions and methods for detection of babesia

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- Number: US 2020/0399716 A1
- Document ID: US20200399716A1
- Status: Published application
- Kind: A1
- Application number: 16/927,191
- Filed: 2020-07-13
- Published: 2020-12-24
- Earliest claimed: 2017-07-18
- Assignees: Roche Molecular Systems, Inc.
- Applicants: Roche Molecular Systems, Inc.
- Inventors: Ellen H. Fiss; Jingtao Sun
- Web page: https://patentgrep.com/patent/US20200399716A1

## CPC

- C12Q 1/6893: for protozoa
- 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

Methods for the rapid detection of the presence or absence of Babesia in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers and probes targeting Babesia and kits are provided that are designed for the detection of Babesia, including, but not limited to, the Babesia species of B. microti, B. divergens, B. duncani, and B. venatorum. Also described are kits, reaction mixtures, and oligonucleotides (e.g., primer and probe) for the amplification and detection of Babesia.

## Claims

### Claim 1

A method of detecting the Babesia species of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum in a sample, the method comprising:
(a) performing an amplification step comprising contacting the sample with one or more set of primers to produce an amplification product, if a target nucleic acid of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum is present in the sample;
(b) performing a hybridization step comprising contacting the amplification product, if the target nucleic acid of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum is present in the sample, with one or more probes; and
(c) detecting the presence or absence of the amplification product, wherein the presence of the amplification product is indicative of the presence of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum in the sample, and wherein the absence of the amplification product is indicative of the absence of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum in the sample; and
wherein the one or more set of primers and the one or more probes comprise:
(i) a set of primers for amplification of B. microti comprising:
a primer comprising the nucleic acid sequence of SEQ ID NO:1, or a complement thereof, and
a primer comprising the nucleic acid sequence of SEQ ID NO:3, or a complement thereof; and
a probe for hybridizing to the amplification product of B. microti comprising the nucleic acid sequence of SEQ ID NO:2, or a complement thereof; and/or
(2) a set of primers for amplification of B. divergens, B. duncani, and/or B. venatorum comprising:
a primer comprising the nucleic acid sequence of SEQ ID NO:4, or a complement thereof, and
one or more primers compromising the nucleic acid sequence(s) of SEQ ID NO:6 or SEQ ID NO:7 or a combination of SEQ ID NOs:6 and 7, or a complement thereof; and
a probe for hybridizing to the amplification product of B. divergens, B. duncani, and/or B. venatorum comprising the nucleic acid sequence of SEQ ID NO:5, or a complement thereof.

### Claim at position 2

2-13. (canceled)

### Claim 14 (depends on 1)

The method of claim 1, wherein the sample is a biological sample.

### Claim 15 (depends on 14)

The method of claim 14, wherein the biological sample is whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, dermal swabs, nasal swabs, wound swabs, blood cultures, skin, or soft tissue infections.

### Claim 16 (depends on 15)

The method of claim 15, wherein the biological sample is whole blood.

### Claim 17 (depends on 1)

The method of claim 1, wherein the hybridization step comprises contacting the amplification product with the one or more probes, wherein the one or more probes is labeled with a donor fluorescent moiety and a corresponding acceptor moiety; and the detecting step comprises detecting the presence or absence of fluorescent resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the one or more probes, wherein the presence or absence of fluorescence is indicative of the presence or absence of Babesia in the sample.

### Claim 18 (depends on 17)

The method of claim 17, wherein the donor fluorescent moiety is HEX or FAM.

### Claim 19 (depends on 18)

The method of claim 18, wherein the donor fluorescent moiety is HEX.

### Claim 20 (depends on 17)

The method of claim 17, wherein the acceptor moiety is a quencher.

### Claim 21 (depends on 20)

The method of claim 20, wherein the quencher is BlackHole Quencher™-2 (BHQ-2).

### Claim 22 (depends on 17)

The method of claim 17, wherein the donor fluorescent moiety and the acceptor moiety are within 5 to 20 nucleotides of each other.

### Claim 23 (depends on 22)

The method of claim 22, wherein the donor fluorescent moiety and the acceptor moiety are within 7 to 10 nucleotides of each other.

### Claim 24 (depends on 23)

The method of claim 23, wherein the donor fluorescent moiety and the acceptor moiety are within 8 nucleotides of each other.

### Claim 25 (depends on 23)

The method of claim 23, wherein the donor fluorescent moiety and the acceptor moiety are within 10 nucleotides of each other.

### Claim 26

A method of detecting Babesia of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum in a sample, wherein the sample is whole blood, the method comprising:
(a) performing an amplification step comprising contacting the sample with one or more set of primers to produce an amplification product, if a target nucleic acid of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum is present in the whole blood sample;
(b) performing a hybridization step comprising contacting the amplification product, if the target nucleic acid of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum is present in the sample, with one or more probes; and
(c) detecting the presence or absence of the amplification product, wherein the presence of the amplification product is indicative of the presence of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum in the sample, and wherein the absence of the amplification product is indicative of the absence of (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B. venatorum in the sample; and
wherein the one or more set of primers and the one or more probes comprise:
(1) a set of primers for amplification of B. microti comprising:
a primer comprising the nucleic acid sequence of SEQ ID NO:1, or a complement thereof; and
a primer compromising the nucleic acid sequence of SEQ ID NO:3, or a complement thereof; and
a probe for hybridizing to the amplification product of B. microti comprising the nucleic acid sequence of SEQ ID NO:2, or a complement thereof; and/or
(2) a set of primers for amplification of B. divergens, B. duncani, and/or B. venatorum comprising:
a primer comprising the nucleic acid sequence of SEQ ID NO:4, or a complement thereof, and
one or more primers compromising the nucleic acid sequence(s) of SEQ ID NO:6 or SEQ ID NO:7 or a combination of SEQ ID NOs:6 and 7, or a complement thereof; and
a probe for hybridizing to the amplification product of B. divergens, B. duncani, and/or B. venatorum comprising the nucleic acid sequence of SEQ ID NO:5, or a complement thereof.

### Claim at position 16

27-64. (canceled)

### Claim 65

A method of detecting the Babesia species of B. microti in a sample, wherein the sample is whole blood, the method comprising:
(a) performing an amplification step comprising contacting the sample with one or more set of primers to produce an amplification product, if a target nucleic acid of B. microti is present in the whole blood sample;
(b) performing a hybridization step comprising contacting the amplification product, if the target nucleic acid of B. microti is present in the sample, with one or more probes; and
(c) detecting the presence or absence of the amplification product, wherein the presence of the amplification product is indicative of the presence of B. microti in the sample, and wherein the absence of the amplification product is indicative of the absence of B. microti in the sample; and
wherein the one or more set of primers and the one or more probes comprise: (1) a set of primers for amplification of B. microti comprising: (i) a primer comprising the nucleic acid sequence of SEQ ID NO:1, or a complement thereof, and (ii) a primer compromising the nucleic acid sequence of SEQ ID NO:3, or a complement thereof; and (2) a probe for hybridizing to the amplification product of B. microti comprising the nucleic acid sequence of SEQ ID NO:2, or a complement thereof.

### Claim 66

A method of detecting the Babesia species of B. divergens, B. duncani, and/or B. venatorum in a sample, wherein the sample is whole blood, the method comprising:
(a) performing an amplification step comprising contacting the sample with one or more set of primers to produce an amplification product, if a target nucleic acid of B. divergens, B. duncani, and/or B. venatorum is present in the whole blood sample;
(b) performing a hybridization step comprising contacting the amplification product, if the target nucleic acid of B. divergens, B. duncani, and/or B. venatorum is present in the sample, with one or more probes; and
(c) detecting the presence or absence of the amplification product, wherein the presence of the amplification product is indicative of the presence of B. divergens, B. duncani, and/or B. venatorum in the sample, and wherein the absence of the amplification product is indicative of the absence of B. divergens, B. duncani, and/or B. venatorum in the sample; and
wherein the one or more set of primers and the one or more probes comprise: (1) a set of primers for amplification of B. divergens, B. duncani, and/or B. venatorum comprising, (i) a primer comprising the nucleic acid sequence of SEQ ID NO:4, or a complement thereof and (ii) one or more primers compromising the nucleic acid sequence(s) of SEQ ID NO:6 or SEQ ID NO:7 or a combination of SEQ ID NOs:6 and 7, or a complement thereof; and (2) a probe for hybridizing to the amplification product of B. divergens, B. duncani, and/or B. venatorum comprising the nucleic acid sequence of SEQ ID NO:5, or a complement thereof.

### Claim 67 (depends on 65, 66)

The method of claim 65 or 66, wherein the sample is a biological sample.

### Claim 68 (depends on 67)

The method of claim 67, wherein the biological sample is whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, dermal swabs, nasal swabs, wound swabs, blood cultures, skin, or soft tissue infections.

### Claim 69 (depends on 68)

The method of claim 68, wherein the biological sample is whole blood.

### Claim 70 (depends on 65, 66)

The method of claim 65 or 66, wherein the hybridization step comprises contacting the amplification product with the one or more probes, wherein the one or more probes is labeled with a donor fluorescent moiety and a corresponding acceptor moiety; and the detecting step comprises detecting the presence or absence of fluorescent resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the one or more probes, wherein the presence or absence of fluorescence is indicative of the presence or absence of Babesia in the sample.

### Claim 71 (depends on 70)

The method of claim 70, wherein the donor fluorescent moiety is HEX or FAM.

### Claim 72 (depends on 71)

The method of claim 71, wherein the donor fluorescent moiety is HEX.

### Claim 73 (depends on 70)

The method of claim 70, wherein the acceptor moiety is a quencher.

### Claim 74 (depends on 73)

The method of claim 73, wherein the quencher is BlackHole Quencher™-2 (BHQ-2).

### Claim 75 (depends on 70)

The method of claim 70, wherein the donor fluorescent moiety and the acceptor moiety are within 5 to 20 nucleotides of each other.

### Claim 76 (depends on 75)

The method of claim 75, wherein the donor fluorescent moiety and the acceptor moiety are within 7 to 10 nucleotides of each other.

### Claim 77 (depends on 76)

The method of claim 76, wherein the donor fluorescent moiety and the acceptor moiety are within 8 nucleotides of each other.

### Claim 78 (depends on 76)

The method of claim 76, wherein the donor fluorescent moiety and the acceptor moiety are within 10 nucleotides of each other.