Rapid detection of infectious agents

• US 10,640,833 B2
• Filed: 06/26/2015
• Issued: 05/05/2020
• 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.
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US15/321,260
• Publication Number:
  US 20170191117A1
• Time in Patent Office: 1,775 Days
• Field of Search: 435 61, 435 611, 435 612, 435 911, 435 912, 435 9151, 435183, 436 94, 436501, 536 231, 536 243, 536 2433, 536 253, 536 2532
• 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 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 different primers specifically hybridizing to said target nucleic acid for generating an amplicon, wherein each of said primers comprise at least 10 contiguous nucleotides selected from SEQ ID NOs;

2-4, 9-10, 12, 14-23, 25-26, and 28-32 and performing an amplification reaction by incubating said nucleic acids with said amplification reagents for a period of time under conditions sufficient to amplify said target nucleic acid; and

b) detecting the target nucleic acid by detecting an amplicon from said amplification reaction;

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:

2-4, 9-10, 12, 14-23, 25-26, and 28-32.

3. The method of claim 1, wherein said amplification reagents further comprise at least one detectable probe specifically hybridizing to a sequence portion of the amplicon.

4. The method of claim 1, wherein said detecting said amplicon in step (b) comprises detecting hybridization of a detectable probe to said 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 a 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 said amplification reaction in step (a) comprises real-time PCR amplification.

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

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

11. The method of claim 1, wherein said amplification reagents further 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 said amplification reaction in step (a) comprises standard PCR.

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

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

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