RAPID DETECTION OF INFECTIOUS AGENTS

• US 20170191117A1
• Filed: 06/26/2015
• Published: 07/06/2017
• Est. Priority Date: 06/26/2014
• Status: Active Grant

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Inventors: RAND, Kenneth H., HOUCK, Herbert J.
• Granted Patent: US 10,640,833 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The invention generally relates to a method for detecting a target nucleic acid in a sample. This invention is useful for detecting bacterial or viral agents in a sample, and is able to detect nucleic acids from a broad variety of, e.g., bacteria, rather than only one or a few different bacteria at a time.

Claims

1. A method for detecting a target nucleic acid that may be present in a sample, said target nucleic acid, said method comprising the steps of:

a) contacting nucleic acids from said sample with amplification reagents comprising a DNA polymerase, nucleotide monomers, and two or more primers for generating an amplicon, wherein said primers comprise at least a 10 contiguous nucleic acids from one or more of SEQ ID NOs;

1-4, 9-10, 12-23, 25-26, and 28-32.and incubating said nucleic acids with said amplification reagents for a period of time and under conditions sufficient to amplify said target nucleic acid; and

b) detecting the presence or absence of said amplicon;

wherein the presence of said amplicon indicates the presence of said target nucleic acid in said sample.

2. The method of claim 1, wherein the primers used in step (a) are selected from the group consisting of SEQ ID NOs:

1-4, 9-10, 12-23, 25-26, and 28-32.

3. The method of claim 1, wherein amplification reagents further comprise at least one detectable probe specific for a sequence portion of the amplicon.

4. The method of claim 1, wherein step (b) comprises using a detectable probe and detecting hybridization of said detectable probe to the amplicon.

5. The method of claim 4, wherein said detectable probe is selected from the group consisting of SEQ ID Nos. 5-8, 11, 24, 27, 33-37, or an effective fragment thereof.

6. The method of claim 1, wherein the sample comprises RNA and the amplification reagents further comprise cleaned reverse transcriptase.

7. The method of claim 1, wherein the primers used in step (a) are selected from the group consisting of SEQ ID NOs:

25, 26, 28, 29, 30, 31, and 32.

8. The method of claim 1, wherein steps step (a) comprises real-time PCR amplification.

9. The method of claim 8, wherein said amplification reagents comprise a sequence-specific DNA probe consisting of oligonucleotides that are labelled with a fluorescent reporter.

10. The method of claim 8, wherein said amplification reagents comprise a non-specific fluorescent dye that intercalates with any double-stranded DNA.

11. The method of claim 1, wherein said amplification reagents comprise a control target nucleic acid.

12. The method of claim 11, wherein said control target nucleic acid is a quantitative control nucleic acid.

13. The method of claim 1, wherein step (a) comprises standard PCR.

14. The method of claim 1, wherein the amplification reagents comprise reverse transcriptase.

15. The method of claim 14, wherein the reverse transcriptase is cleaned reverse transcriptase.

16. The method of claim 15, wherein the cleaned reverse transcriptase is produced by enzymatic, chemical, or physical treatment of contaminated reverse transcriptase.

17. A kit for performance of the method of claim 1, which comprises amplification reagents comprising a DNA polymerase, nucleotide monomers, and two or more primers for generating an amplicon, wherein said primers comprise at least a 10 contiguous nucleic acids from one or more of SEQ ID NOs:

1-4, 9-10, 12-23, 25-26, and 28-32.

18. The kit of claim 17 which further comprises a probe, wherein said probe is selected from the group consisting of SEQ ID Nos. 5-8, 11, 24, 27, 33-37,

19. The kit of claim 18, wherein said probe is a detectable probe.

20. The kit of claim 17 which further comprises a cleaned reverse transcriptase.

21. The kit of claim 17 which further comprises a control nucleic acid.