IP Library › Granted Patent US 12,264,369
Granted Patent B2
US 12,264,369 · App. 17/485,953 · Granted Apr 1, 2025

Real-time PCR for the detection of pathogens

Inventors: Bernard J. Wolff (Roswell, GA); Jonas M. Winchell (Lilburn, GA); Maureen Diaz (Atlanta, GA)
Assignee: The Government of the United States of America as represented by the Secretary of the Deptartment of Health and Human Services
C12Q1/689C12Q1/6895C12Q2600/158C12Q2600/16
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Quick Facts
Patent No.
US 12,264,369
App. No.
17/485,953
Granted
Apr 1, 2025
Kind
B2
Abstract

Methods for detecting presence of one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and Neisseria meningitidis nucleic acids in a sample, such as a biological sample obtained from a subject, or an environmental sample, are provided. This disclosure also provides probes, primers, and kits for detecting one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and Neisseria meningitidis in a sample.

Claims (55)

1. A method for detecting presence of Klebsiella pneumoniae in a sample, comprising:

contacting the sample with a nucleic acid probe comprising the nucleic acid sequence of SEQ ID NO: 60, or the reverse complement thereof, and a donor fluorophore, an acceptor fluorophore, or a combination thereof;

amplifying a nucleic acid from Klebsiella pneumoniae, wherein the amplifying comprises contacting the sample with a pair of primers comprising the nucleic acid sequences of SEQ ID NOs: 58 and 59; and

detecting hybridization between the nucleic acid probe and a nucleic acid in the sample, wherein detection of hybridization indicates the presence of Klebsiella pneumoniae in the sample.

2. The method of claim 1 , further comprising detecting Ureaplasma spp., in the sample by:

(a) contacting the sample with

a probe comprising or consisting of the nucleic acid sequence set forth as SEQ ID NO: 47; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Ureaplasma spp. in the sample.

3. The method of claim 2 , further comprising amplifying a nucleic acid from Ureaplasma spp. by contacting the nucleic acid with at least one primer,

wherein the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46.

4. The method of claim 1 , wherein the sample comprises a biological sample or environmental sample.

5. The method of claim 4 , wherein the sample is a biological sample comprising tissue, blood, serum, cerebral spinal fluid, middle ear fluid, bronchoalveolar lavage, tracheal aspirate, sputum, nasopharyngeal aspirate, oropharyngeal aspirate, or saliva or the sample is an environmental sample comprising a food sample, a water sample, or a surface swab.

6. An isolated nucleic acid probe consisting of the nucleic acid sequence of SEQ ID NO: 60 and a donor fluorophore, an acceptor fluorophore, or a combination thereof.

7. A kit for detection of Klebsiella pneumoniae, comprising the isolated nucleic acid probe of claim 6 and a pair of primers comprising the nucleic acid sequences of SEQ ID NOs: 58 and 59.

8. The kit of claim 7 , further comprising one or more primers comprising or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 45 and 46.

9. The kit of claim 7 , further comprising an isolated nucleic acid probe comprising the nucleic acid sequence of SEQ ID NO: 47, and a donor fluorophore, an acceptor fluorophore, or a combination thereof.

10. The method of claim 1 , further comprising detecting Acinetobacter baumannii in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 14; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Acinetobacter baumannii in the sample.

11. The method of claim 1 , further comprising detecting Pseudomonas aeruginosa in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 17; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Pseudomonas aeruginosa in the sample.

12. The method of claim 1 , further comprising detecting Staphylococcus aureus in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 32; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Staphylococcus aureus in the sample.

13. The method of claim 1 , further comprising detecting Toxoplasma gondii in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 23; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Toxoplasma gondii in the sample.

14. The method of claim 1 , further comprising detecting Moraxella catarrhalis in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 26; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Moraxella catarrhalis in the sample.

15. The method of claim 1 , further comprising detecting Escherichia coli and/or Shigella in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Escherichia coli and/or Shigella in the sample.

16. The method of claim 1 , further comprising detecting Chlamydia trachomatis in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 38; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Chlamydia trachomatis in the sample.

17. The method of claim 1 , further comprising detecting Ureaplasma urealyticum in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 41; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Ureaplasma urealyticum in the sample.

18. The method of claim 1 , further comprising detecting Ureaplasma parvum in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 44; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Ureaplasma parvum in the sample.

19. The method of claim 1 , further comprising detecting Streptococcus agalactiae in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 56; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Streptococcus agalactiae in the sample.

20. The method of claim 1 , further comprising detecting Bartonella spp. in the sample by:

(a) contacting the sample with a nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 50; and

(b) detecting hybridization between the probe and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of Bartonella spp. in the sample.

21. A method for detecting presence of Klebsiella pneumoniae in a sample, comprising:

contacting the sample with a nucleic acid probe comprising the nucleic acid sequence of SEQ ID NO: 20, or the reverse complement thereof, and a donor fluorophore, an acceptor fluorophore, or a combination thereof;

amplifying a nucleic acid from Klebsiella pneumoniae , wherein the amplifying comprises contacting the sample with a pair of primers comprising the nucleic acid sequences of SEQ ID NOs: 18 and 19; and

detecting hybridization between the nucleic acid probe and a nucleic acid in the sample, wherein detection of hybridization indicates the presence of Klebsiella pneumoniae in the sample.

22. An isolated nucleic acid probe consisting of the nucleic acid sequence of SEQ ID NO: 20 and a donor fluorophore, an acceptor fluorophore, or a combination thereof.

23. A kit for detection of Klebsiella pneumoniae, comprising the isolated nucleic acid probe of claim 22 and a pair of primers comprising the nucleic acid sequences of SEQ ID NOs: 18 and 19.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 27, 2021
From: WOLFF, BERNARD
To: THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
Reel/Frame 057609/0519 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 27, 2021
From: WINCHELL, JONAS M.
To: THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
Reel/Frame 057609/0564 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 27, 2021
From: DIAZ, MAUREEN
To: THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
Reel/Frame 057609/0596 →
Continuity (4)
Division 16056147 · Aug 6, 2018
Division 14398390
Provisional Application 61642091 · May 3, 2012
Related Publication 20220010365A1 · Jan 13, 2022
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