Bioagent detection oligonucleotides

• US 9,970,061 B2
• Filed: 12/27/2012
• Issued: 05/15/2018
• Est. Priority Date: 12/27/2011
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Ecker, David J., Hofstadler, Steven A., Sampath, Rangarajan, Blyn, Lawrence B., Hall, Thomas A., Eshoo, Mark W.
• Primary Examiner(s): GOLDBERG, JEANINE ANNE
• Application Number:
  US14/369,618
• Publication Number:
  US 20150184231A1
• Time in Patent Office: 1,965 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The present invention compositions, methods and systems to identify, detect, and/or quantify bacterial DNA in the presence of contaminating non-bacterial DNA. In particular, the present invention provides oligonucleotides configured to detect a relatively small amount of bacterial DNA in the presence of an overwhelmingly large amount of contaminating human DNA.

Claims

1. A method of detecting bacterial DNA in the presence of contaminating human DNA comprising:

a) contacting a sample comprising human genomic DNA, contaminants, and possibly bacterial DNA with an oligonucleotide wherein said oligonucleotide is 1 a sequence selected from SEQ ID NOs;

1-18;

b) allowing said oligonucleotide to hybridize to bacterial DNA, if bacterial DNA is present in said sample; and

c) detecting said bacterial DNA in the presence of contaminating human DNA, if bacterial DNA is present in said sample, based upon hybridization of said oligonucleotide to said bacterial DNA.

2. The method of claim 1, further comprising quantifying said bacterial DNA.

3. The method of claim 1, further comprising using SCODA to separate said bacterial DNA from said human genomic DNA.

4. The method of claim 1, wherein said oligonucleotide comprises a label.

5. The method of claim 4, wherein said label is a fluorescent label, a luminescent label, a chemiluminescent label, radioactive label, a quencher label, an interacting label, or a mass-tagged label.

6. The method of claim 1, wherein said oligonucleotide is a capture oligonucleotide immobilized in a SCODA gel.

7. The method of claim 6, wherein said bacterial DNA is eluted from said SCODA gel and amplified with a primer pair comprising a sequence selected from SEQ ID NOs:

1-18 prior to detection.

8. The method of claim 6, wherein said bacterial DNA is amplified in said SCODA gel using in situ PCR methods with a primer pair comprising a sequence selected from SEQ ID NOs:

1-18 prior to detection, or prior to elution and detection.

9. The method of claim 8, wherein an electrical field, a magnetic field, a flow field, or combination thereof promotes hybridization and disassociation of said bacterial DNA and said immobilized primers during rounds of said PCR.

10. The method of claim 8, wherein said bacterial DNA is detected without elution from said SCODA gel wherein a sequence of said primer pair is detectably labeled.

11. The method of claim 7, wherein said detection comprises next-generation sequencing detection.

12. The method of claim 7, wherein said detection comprises quantifying said bacterial DNA.