Rapid and comprehensive identification of prokaryotic organisms

• US 20080261222A1
• Filed: 01/25/2008
• Published: 10/23/2008
• Est. Priority Date: 01/25/2007
• Status: Active Grant

• Current Assignee: Peter K. Rogan
• Original Assignee: ThinkVillage Corporation
• Inventors: Rogan, Peter K.
• Granted Patent: US 8,076,104 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

An improved method for rapid identification of microorganisms is disclosed, along with sequences of PCR primers optimized for this purpose. The primers are designed based on information analysis of sequences from a large number of organism to amplify certain segments of genomic DNA whose sequences are unique among different organisms. The PCR products are compared with a DNA sequence database to obtain the identity of the microorganisms. This approach provides an accurate and fast identification and taxonomic assignment of microbial species.

Claims

1. A pair of oligonucleotides to be used as primers for DNA amplification, comprising:

4. The use of the primer pairs recited in any one of claims 1, 2, and 3 to amplify the DNA of an organism.

2. A pair of oligonucleotides to be used as primers for DNA amplification, comprising:

3. A pair of oligonucleotides to be used as primers for DNA amplification, comprising:

5. A method for obtaining data for taxonomic assignment of unknown species comprising:

(a) searching for a divergent segment of DNA with low average information content determined quantitatively surrounded by two conserved segments of said DNA with high average information content determined quantitatively;

(b) designing primers for PCR amplification of said divergent segment by constructing a sequence logo for said DNA such that said primers contain a set of sequences present in said sequence logo that encompass the nucleotide variability of said conserved segments, which primers can anneal to said conserved segments;

(c) amplifying said divergent segment of DNA by PCR technique using said primers to obtain PCR products;

(d) separating said PCR products; and

(e) characterizing the separated PCR products based upon properties selected from the group consisting of nucleotide composition, base composition, nucleotide sequence, DNA structure, mass ratio of the DNA molecules or fragments derived therefrom, chemical reactivity of the DNA molecules, binding properties to other DNA molecules or proteins, thermal stability, and combination thereof.

6. The method of claim 5, wherein the searching for said divergent segment of DNA surrounded by said conserved segments is performed in the region of the DNA coding for the ribosomal RNA of any organism.

7. The method of claim 6, wherein said ribosomal RNA is the 28S ribosomal RNA from eukaryotic organisms or the 16S ribosomal RNA from prokaryotic organisms.

8. The method of claim 7, wherein the separated PCR products are characterized based upon nucleotide sequence.

9. The method of claim 8, wherein sequences of the separated PCR products are determined by mass spectrometry.

10. The method of claim 7, wherein the PCR products are separated by means selected from the group consisting of magnetic separation, gel purification, mass spectrometry, and combination thereof.

11. The method of claim 10, wherein the PCR products are separated by gel purification.

12. The method of claim 10, wherein the PCR products are separated by denaturing gel electrophoresis.

13. The method of claim 7, wherein steps (d) and (e) are accomplished by mass spectrometry.

14. A method for identifying an organism in a sample, comprising:

(a) searching for a divergent segment of DNA with low average information content determined quantitatively surrounded by two conserved segments of said DNA with high average information content determined quantitatively;

(b) designing primers for PCR amplification of said divergent segment by constructing a sequence logo for said DNA such that said primers contain a set of sequences present in said sequence logo that encompass the nucleotide variability of said conserved segments, which primers can anneal to said conserved segments;

(c) amplifying said divergent segment of DNA by PCR technique using said primers to obtain PCR products;

(d) separating said PCR products; and

(e) characterizing the separated PCR products based upon properties selected from the group consisting of nucleotide composition, base composition, nucleotide sequence, DNA structure, mass ratio of the DNA molecules or fragments derived therefrom, chemical reactivity of the DNA molecules, binding properties to other DNA molecules or proteins, thermal stability, and combination thereof.

15. The method of claim 14, further comprising a sample processing step to be performed before step (c), said sample processing step being selected from the group consisting of lysis of red blood cells, removal or addition of salts, concentration of samples and dilution of samples.

16. The method of claim 14, further comprising a step wherein the existence of a pathological condition or a disease in an individual is determined base upon the identity of said organism obtained in steps (a)-(e).

17. The method of claim 14, wherein the searching for said divergent segment of DNA surrounded by said conserved segments is performed in the region of the DNA coding for the ribosomal RNA of any organism.

18. The method of claim 17, wherein said ribosomal RNA is the 28S ribosomal RNA from eukaryotic organisms or the 16S ribosomal RNA from prokaryotic organisms.

19. The method of claim 18, wherein the separated PCR products are characterized based upon nucleotide sequence.

20. The method of claim 19, wherein sequences of the separated PCR products are determined by mass spectrometry.

21. The method of claim 20, wherein the PCR products are separated by means selected from the group consisting of magnetic separation, gel purification, mass spectrometry, and combination thereof.

22. The method of claim 21, wherein the PCR products are separated by gel purification.

23. The method of claim 21, wherein the PCR products are separated by denaturing gel electrophoresis.

24. The method of claim 18, wherein steps (d) and (e) are accomplished by mass spectrometry.

25. The method of claim 16, wherein said organism is from at least one species selected from the group consisting of Acinetobacter, Actinobacillus, Actinomyces pyogenes, Actinomyces pyogenes, Aeromonas hydrophilia, Alcaligenes fecalis, Aligella urethralis, Alteromonas putriaciens, Alteromonas putrifuciens, Bacteriodes distasonis, Bacteriodes fragila, Bacteriodes melaminogenicus, Bacteriodes ovatus, Bacteriodes thetaiomicion, Bacteriodes uniformis, Campylobacter, Candida albicans, Capnocytophaga, CDC group Ivc, Chlamydia trachomatis, Citrobacter fruendii, Clostridium difficle, Clostridium histolyticum, Clostridium perfingens, Clostridium septicum, Clostridium sordelli, Clostridium sporogenes, Corynebacterium diptheria, Corynebacterium pseudodoi, E. coli, E. coli 0157-H7, E. coli-β

lactamase positive, Enterobacter aerogenes, Enterobacter cloecae, Enterobacter fecalis, Eubacterium lentum, Flavobacterium meningiosepticum, Fusobacterium, Fusobacterium miningio, Haemophilia parainfluenza, Haemophilis influenza, Haemophilus aphrophilus, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella rhinoscleromatis, Legionella micdliea, Legionella pneumophilia, Leuconostoc lactic, Leuconostoc mesanteriodas, Listeria murrayi, Mima, Mycobacterium avium intracellulari, Mycobacterium flavescens, Mycobacterium gordoniae, Mycobacterium terra group, Mycobacterium tuberculoses, Neisseria cinerea, Neisseria gonorrhea, Neisseria lactamica, Neisseria meningiditis, Neisseria sicca, Nocardia brasiliensis, Pasteurella multocida, Proteus mirabilis, Proteus vulgaris, Pseudomonas cepatia (strain I), Pseudomonas cepatia (strain II), Salmonella cholerasuis, Salmonella dublin, Salmonella muenchen, Salmonella paratyphi, Salmonella typhimurium, Serratia oderifera, Shigella boydii, Shigella dysenteriae, Shigella felxneri, Shigella sonneri, Staphylococcus aureus, Staphylococcus epi., Staphylococcus saphrophy., Streptococcus alpha, Streptococcus beta (Group C), Streptococcus beta (Group F), Streptococcus bovis, Streptococcus fecalis, Streptococcus Group B, Streptococcus mitis, Streptococcus mutans, Streptococcus pneumonia, Torulopsis globrata, Treponema denticola, Treponema pallidum, Treponema pertenue, Treponema phagedenis, Treponema refrigens, Vibro paruhen, and Yersinia enterolitica.