Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-05
2003-01-28
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S029000, C435S032000, C435S034000, C435S091200, C536S025320, C536S025400
Reexamination Certificate
active
06511804
ABSTRACT:
BACKGROUND OF THE INVENTION
For decades, scientists have attempted to improve the control of infection and the detection of microorganisms. Determining the susceptibility of microorganisms to particular chemical agents has particular importance in clinical medicine, where the practitioner needs to determine not only which of a number of possible microorganisms might be causing the patient's infection, but also to which agents the particular strain infecting the patient is susceptible. This effort may be complicated or even confounded by the presence of normal flora (bacteria which are usually present at certain body locations, but which do not cause disease under normal conditions) or of other microorganisms which happen to be present but which are not the causative agent of the patient's primary disease. Thus, the determination of both the identity of the organisms present in a sample, and the viability of the target organism, is important. As a practical matter, however, the practitioner often cannot risk a decline in the patient's condition while the organisms present are cultured, identified, and tested for antibiotic susceptibility by conventional techniques, and usually selects a broad spectrum agent in the hope it will be effective against the microorganism actually causing the disease.
A number of techniques are known in the art for determining the viability of microorganisms. These techniques generally permit replication of any microorganisms present and therefore require the use of isolated, pure cultures to give meaningful results. For example, U.S. Pat. No. 4,416,995 teaches a method of determining sensitivity of bacteria to chemical agents by measuring the amount of tritiated thymidine which is incorporated into bacteria in the presence of the agent compared to that incorporated into a second culture in the absence of the agent. In U.S. Pat. No. 4,812,394, Dolbeare et al. teach a non-radioactive technique using the simultaneous measurement by flow cytometry of total cellular DNA and incorporated nucleoside analogs. Cells are permitted to grow in the presence of a nucleoside analog, such as 5-bromodeoxyuridine (“BrdU”), and a portion of the double stranded DNA is then made single stranded. That portion is then “stained” by immunochemical means, such as by antibodies to the nucleoside analog. The unaltered, double stranded DNA is then detected by a stain specific for the unaltered DNA, such as the intercalcating dyes propidium iodide or ethidium bromide.
Antibodies to many nucleotide analogs are commercially available to detect incorporation. ELISA kits are commercially available to measure BrdU uptake into DNA as part of cell viability or proliferation assays. For example, Boehringer Mannheim's “Cell Proliferation ELISA, BrdU” incubates a sample with BrdU. DNA in the sample is then fixed to the wells of a microtiter plate and anti-BrdU antibody-peroxidase conjugate is introduced. The presence of BrdU-labeled DNA is then detected calorimetrically by introduction of tetramethylbenzidine (“TMB”) substrate. These methods do not permit the identification of the microorganism, which can be a concern in dealing with the mixed cultures of pathogenic microorganisms and normal microbial flora which are often obtained from patients in clinical situations.
Finally, amplification methods for nucleic acids are known in the art. These methods have the disadvantage of amplifying everything present in a sample. They therefore amplify the nucleic acids of both living and dead microorganisms present in a sample and cannot be used to determine viability of the microorganisms.
SUMMARY OF THE INVENTION
The subject invention discloses an assay for determining the identity of live microorganisms in mixed cultures comprising the steps of (i) culturing the microorganism in a medium comprising a nucleic acid analog recognized by an analog-specific binding member, which is incorporated into nucleic acids of replicating cells of the microorganism, (ii) lysing the microorganism to release its nucleic acids, (iii) capturing nucleic acids incorporating the analog using the analog-specific binding member, (iv) separating the captured nucleic acids from those which were not captured, (v) amplifying the captured nucleic acids using methods selective for particular organisms or groups of organisms, and (vi) detecting the presence or absence of amplified, captured nucleic acids. The amplification can be by polymerase chain reaction.
The invention further discloses that the assay can be used where the microorganism is a pathogen, where the pathogen is a pathogen of a mammal, and where the microorganism is a bacterium, a virus, a fungus, or a protozoan.
Further, the invention can be used to determine the sensitivity of microorganisms in the culture to chemical agents without needing first to isolate the microorganisms.
The invention discloses that the nucleic acid analog can be bromodeoxyuridine. Additionally, the invention discloses that the binding member can be an antibody. Moreover, the analog-binding member can be attached to a solid support.
The invention can further be practiced by repeating the steps at least once (or dividing the medium containing the microorganisms into two aliquots) wherein one of the series of steps (or one of the aliquots) has an antibiotic added to the medium and at least one of the series of steps (or one of the aliquots) does not have an antibiotic added to the medium.
Finally, the invention discloses kits for determining the identity of microorganisms in a mixed culture, or the ability of selected organisms in a mixed culture to replicate, or both, comprising a DNA or RNA analog (depending on the nature of the microorganism of interest) which is recognized by an analog-specific binding member and which is permissive of incorporation into nucleic acids of the microorganism, an analog-specific binding member for capturing nucleic acids incorporating the analog, and instructions for using these components in an assay for determining the ability of microorganisms of interest to replicate.
REFERENCES:
patent: 4416995 (1983-11-01), Amaral
patent: 4780406 (1988-10-01), Dolbeare et al.
patent: 4812394 (1989-03-01), Dolbeare et al.
patent: 5648227 (1997-07-01), Basboll
Jou, N-T. et al. J. Clin. Microbiol. 35(5):1161-1165 (May 1997).*
Kell, W. M. et al. Meth. Mol. Cell. Biol. 5(2):105-111 (1995).*
Ahern, H. The Scientist 9(15):20 (Jul. 1995).*
Boehringer Mannheim Catalog “Cell Biology and Immunochemistry: Cell Proliferation, Cytotoxicity, and Cell Death,” Chapter 2, Catalog Nos. 1 647 229 & 1 585 045, pp. 213-222.
Haydock Paul V.
Uren Jack R.
Johannsen Diana
Myers Carla J.
Saigene Corporation
Townsend and Townsend / and Crew LLP
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