COMPOSITIONS AND METHODS FOR DETECTION OF TRICHOMONAS VAGINALIS

• US 20170342508A1
• Filed: 05/22/2017
• Published: 11/30/2017
• Est. Priority Date: 05/27/2016
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Harris, Jody, Lu, Shi-Da Y., Mangipudi, Kalyani, Sun, Jingtao
• Granted Patent: US 10,934,597 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6893 for protozoa
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Methods for the rapid detection of the presence or absence of Trichomonas vaginalis (TV) 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, probes targeting the target TV gene, along with kits are provided that are designed for the detection of TV.

Claims

1. A method of detecting Trichomonas vaginalis (TV) in a sample, the method comprising:

performing an amplifying step comprising contacting the sample with a set of target TV gene primers to produce an amplification product if a target TV gene nucleic acid is present in the sample;

performing a hybridizing step comprising contacting the amplification product with one or more detectable target TV gene probes; and

detecting the presence or absence of the amplification product, wherein the presence of the amplification product is indicative of the presence of TV in the sample and wherein the absence of the amplification product is indicative of the absence of TV in the sample;

wherein the set of target TV gene primers comprise a first primer comprising a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs;

1-9, 19-28, 45-48, 55-64, and 80-89 or a complement thereof, and a second primer comprising a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs;

10-13, 29-38, 49-52, 65-74, and 90-99, or a complement thereof; and

wherein the one or more detectable target TV gene probes comprises a third oligonucleotide sequence selected from the group consisting of SEQ ID NOs;

14-18, 39-44, 53-54, 75-79, and 100-105, or the complement thereof.

2. The method of claim 1, wherein:

the hybridizing step comprises contacting the amplification product with the detectable target TV gene probe that is labeled with a donor fluorescent moiety and a corresponding acceptor moiety; and

the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, wherein the presence or absence of fluorescence is indicative of the presence or absence of TV in the sample.

3. The method of claim 2, wherein said amplifying step employs a polymerase enzyme having 5′

to 3′

nuclease activity.

4. The method of claim 2, wherein the donor fluorescent moiety and the corresponding acceptor moiety are within no more than 8-20 nucleotides of each other on the probe.

5. The method of claim 2, wherein the acceptor moiety is a quencher.

6. A kit for detecting a nucleic acid of Trichomonas vaginalis (TV) comprising:

a first primer comprising a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs;

1-9, 19-28, 45-48, 55-64, and 80-89, or a complement thereof;

a second primer comprising a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs;

10-13, 29-38, 49-52, 65-74, and 90-99, or a complement thereof; and

a fluorescently detectably labeled probe comprising a third oligonucleotide sequence selected from the group consisting of SEQ ID NOs;

14-18, 39-44, 53-54, 75-79, and 100-105, or a complement thereof, the detectably labeled probe configured to hybridize to an amplicon generated by the first primer and the second primer.

7. The kit of claim 6, wherein the third detectably labeled oligonucleotide sequence comprises a donor fluorescent moiety and a corresponding acceptor moiety.

8. The kit of claim 7, wherein the acceptor moiety is a quencher.

9. The kit of claim 6, further comprising at least one of nucleoside triphosphates, nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase.

10. The kit of claim 6, wherein at least one of the first, second, and third oligonucleotides comprises at least one modified nucleotide.

11. The kit of claim 6, wherein the first, second, and third oligonucleotides have 40 or fewer nucleotides.