| Title: |
Ratiometric Pre-rRNA Analysis |
| Document Number: |
20120094285 |
| Publication Date: |
April 19, 2012 |
| Appl. No: |
13/133889 |
| Application Filed: |
December 10, 2009 |
| Abstract: |
Disclosed are compositions and methods for detecting the presence of viable cells in a sample. Included are compositions and methods for increasing the sensitivity of a nucleic acid amplification test for determining the presence of at least one target microorganism in a sample. Also disclosed are compositions and methods for detecting ribosomal RNA precursors (pre-rRNA) as dynamic indicators of viable microorganisms in a sample. |
| Inventors: |
Cangelosi, Gerard A (Seattle, WA, US); Meschke, John Scott (Bothell, WA, US); Weigel, Kris (Seattle, WA, US) |
| Assignees: |
Seattle Biomedical Research Institute (Seattle, WA, US), University of Washington (Seattle, WA, US) |
| Claim: |
1. A method of detecting viable microorganisms in a sample, the method comprising: collecting a sample; nutritionally stimulating a first aliquot of the sample; maintaining a second aliquot of the sample under non-nutritionally stimulating control conditions; incubating the first aliquot and the second aliquot; comparing the level of at least one target pre-rRNA from at least one target microorganism from the first aliquot with the level of the at least one target pre-rRNA from at least one target microorganism in the second aliquot; wherein the ratio of the level of the at least one target pre-rRNA in the first aliquot and the level of the at least one target pre-rRNA in the second aliquot is indicative of viable microorganisms in the sample. |
| Claim: |
2. The method of claim 1, further comprising extracting RNA from the first aliquot and extracting RNA from the second aliquot; and quantifying the level of the at least one target pre-rRNA in the first aliquot and quantifying the level of the at least one target pre-rRNA the second aliquot. |
| Claim: |
3. The method of claim 1, wherein the percentage of the at least one target microorganism that are viable in the sample is at least one of approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.5%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. |
| Claim: |
4. The method of claim 2, wherein extracting the RNA from the first aliquot and the second aliquot and quantifying the at least one pre-rRNA from the first aliquot and the second aliquot comprises the use of a microfluidic device. |
| Claim: |
5. The method of claim 1, further comprising an immunoseparation process wherein the sample is screened for the presence of a particular isolate of the at least one target microorganism in the sample. |
| Claim: |
6. The method of claim 5, wherein the immunoseparation process comprises screening the sample for the presence of E. coli 0157. |
| Claim: |
7. The method of claim 1, wherein the at least one target microorganism is a member of a genera of microorganisms selected from the group consisting of Acinetobacter, Actinobacillus, Aeromonas, Arcobacter, Bacteroides, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Citrobacter, Cronobacter, Edwardsiella, Enterobacter, Escherichia, Eubacterium, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Moraxella, Morganella, Neisseria, Pasteurella, Plesiomonas, Porphyromonas, Prevotella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Stenotrophomonas, Treponema, Veillonella, Vibrio, Yersinia, Actinomyces, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Listeria, Micrococcus, Mobiluncus, Mycobacterium, Nocardia, Peptostreptococcus, Propionibacterium, Rhodococcus, Staphylococcus, Streptococcus, and Streptomyces. |
| Claim: |
8. The method of claim 1, wherein the at least one target microorganism is a Mycobacterium species. |
| Claim: |
9. The method of claim 1, wherein the at least one target microorganism is Aeromonas hydrophila. |
| Claim: |
10. The method of claim 1, wherein the at least one target microorganism is at least one of Chlamydia trachomatis, Legionella pneumonia, Listeria monocytogenes, Campylobacter jejuni, Clostridium difficile, Bacillus anthracis, Francisella tularensis, Rickettsia prowasekii, Rickettsia typhi, and Helicobacter pylori. |
| Claim: |
11. The method of claim 1, wherein the first aliquot is incubated for a time period that is less than the doubling time of the at least one target microorganism. |
| Claim: |
12. The method of claim 1, wherein nutritionally stimulating the first aliquot comprises enriching the first aliquot with nutrients to encourage an upshift of pre-rRNA production in the at least one target microorganism. |
| Claim: |
13. The method of claim 1, wherein the ratio of the level of the at least one target pre-rRNA in the first aliquot and the level of the at least one target pre-rRNA in the second aliquot is ≧1. |
| Claim: |
14. A method of increasing the sensitivity of a nucleic acid amplification test for determining the presence of at least one target microorganism in a sample, wherein the results of a standard (non-ratiometric) nucleic acid amplification test for the sample might be inconclusive for the presence of the at least one target microorganism, the method comprising: dividing the sample into at least a first aliquot and a second aliquot; nutritionally stimulating the first aliquot of the sample; maintaining the second aliquot of the sample under non-nutritionally stimulating control conditions; incubating the first aliquot and the second aliquot; extracting RNA from the first aliquot and extracting RNA from the second aliquot; quantifying the level of at least one target pre-rRNA from the first aliquot; quantifying the level of the at least one target pre-rRNA from the second aliquot; comparing the level of the at least one target pre-rRNA in the first aliquot with the level of the at least one target pre-rRNA in the second aliquot; wherein the ratio of the level of the at least one target pre-rRNA in the first aliquot and the level of target pre-rRNA in the second aliquot is used to improve confidence in borderline results, and thereby to increase the sensitivity of the nucleic acid amplification test for the presences of the at least one microorganism in the sample. |
| Claim: |
15. The method of claim 14, wherein when the ratio of the level of the at least one target pre-rRNA in the first aliquot and the level of target pre-rRNA in the second aliquot is >1, the presence of the at least one target microorganism is confirmed in the sample. |
| Claim: |
16. The method of claim 14, wherein the primary nucleic acid amplification test is inconclusive because it generates a cycle threshold value of >30. |
| Claim: |
17. The method of claim 14, wherein the sample is divided into at least a first aliquot and a second aliquot concurrently with a separate nucleic acid amplification test. |
| Claim: |
18. The method of claim 14, wherein the sample is divided into at least a first aliquot and a second aliquot after completion of the primary nucleic acid amplification test. |
| Claim: |
19. The method of claim 14, wherein the nucleic acid amplification test is a DNA amplification test. |
| Claim: |
20. The method of claim 14, wherein the percentage of the at least one target microorganism that are viable in the sample is at least one of approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.5%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. |
| Claim: |
21. The method of claim 14, wherein extracting the RNA from the first aliquot and the second aliquot and quantifying the at least one pre-rRNA from the first aliquot and the second aliquot comprises the use of a microfluidic device. |
| Claim: |
22. The method of claim 14, further comprising an immunoseparation process wherein the sample is screened for the presence of a particular isolate of the at least one target microorganism in the sample. |
| Claim: |
23. The method of claim 14, wherein the at least one target microorganism is a member of a genera of microorganisms selected from the group consisting of Acinetobacter, Actinobacillus, Aeromonas, Arcobacter, Bacteroides, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Citrobacter, Cronobacter, Edwardsiella, Enterobacter, Escherichia, Eubacterium, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Moraxella, Morganella, Neisseria, Pasteurella, Plesiomonas, Porphyromonas, Prevotella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Stenotrophomonas, Treponema, Veillonella, Vibrio, Yersinia, Actinomyces, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Listeria, Micrococcus, Mobiluncus, Mycobacterium, Nocardia, Peptostreptococcus, Propionibacterium, Rhodococcus, Staphylococcus, Streptococcus, and Streptomyces. |
| Claim: |
24. The method of claim 14, wherein the at least one target microorganism is a Mycobacterium species. |
| Claim: |
25. The method of claim 14, wherein the at least one target microorganism is Aeromonas hydrophila. |
| Claim: |
26. The method of claim 14, wherein the at least one target microorganism is at least one of Chlamydia trachomatis, Legionella pneumonia, Listeria monocytogenes, Campylobacter jejuni, Clostridium difficile, Bacillus anthracis, Francisella tularensis, Rickettsia prowasekii, Rickettsia typhi, and Helicobacter pylori. |
| Claim: |
27. The method of claim 14, wherein the first aliquot is incubated for a time period that is less than the doubling time of the at least one target microorganism. |
| Claim: |
28. The method of claim 14, wherein nutritionally stimulating the first aliquot comprises enriching the first aliquot with nutrients to encourage an upshift of pre-rRNA production in the at least one target microorganism. |
| Claim: |
29. The method of claim 14, wherein the ratio of the level of the at least one target pre-rRNA in the first aliquot and the level of the at least one target pre-rRNA in the second aliquot is ≧1. |
| Current U.S. Class: |
435/611 |
| Current International Class: |
12 |
| Accession Number: |
edspap.20120094285 |
| Database: |
USPTO Patent Applications |
