Detection of multiple nucleic acid sequences in a fluid sample

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage

Reexamination Certificate

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C435S006120, C435S091100, C436S094000

Reexamination Certificate

active

06352827

ABSTRACT:

The present invention relates to the field of assay for multiple nucleic acid sequence analytes in a single fluid sample. More particularly, the present invention is directed to detection, in a sample, of multiple nucleic acid sequence analytes, for example, those characteristic of pathogenic organisms or of oligonucleotide therapeutic agents, as an aid to diagnosis and treatment of disease states, or those characteristic of contaminants in reagents that are required to be sterile.
BACKGROUND OF THE INVENTION
Nucleic acid sequence-based assays can directly or indirectly measure the level of a analyte nucleic acid present in biological fluids. Many such assays are known, for example, as disclosed in U.S. Pat. Nos. 5,124,246; 4,775,619; 5,328,825; 5,232,829; and 5,283,174. Such assays generally rely on amplification, either of the nucleic acid analyte itself or of the signal generated by a probe. The amplification requirement inherent to currently available DNA sequence-based assays necessitates specialized equipment and training of laboratory personnel. When the nucleic acid analyte is amplified, as by the polymerase chain reaction, standardized results are generally not available, and the procedure is susceptible to contamination. Moreover, PCR amplification products often contain sequence inaccuracies. When the signal is amplified, high backgrounds can result from nonspecific binding of the probe to non-analyte nucleic acids and to other species present in the sample.
Occasionally a single biological sample contains more than one clinically relevant nucleic acid analyte. For example, when any of a number of pathogens could cause similar symptomatology, or when an individual may be infected with more than one pathogen, separate assays must be performed for each pathogen. Oligonucleotide-based therapeutic agents are also used in clinical trials, and ultimately, an individual treated with a combination of such agents will require monitoring for their presence.
Using currently available methodologies, a separate assay must be performed for each nucleic acid analyte. The amount of biological sample required for such multiple assays is greater than that required for a single assay, necessitating increased handling, storage, and instrumentation in addition to potential increased patient discomfort. The requirement for separate assays for each nucleic acid analyte can increase the expense of diagnosis as well as the time involved in reaching diagnosis and thus in deciding on appropriate treatment.
Significant difficulties arise in using known nucleic acid sequence-based assays for simultaneous detection of more than one analyte nucleic acid in a biological sample. The presence of more than one probe sequence could result in cross-hybridization to PCR products that contain sequence inaccuracies. The high background problem in signal-amplification assays could interfere with detection of multiple signals. Thus a need exists for novel DNA-based diagnostics which accurately and efficiently detect multiple nucleic acid analytes, with minimal expense and inconvenience for clinician and patient.
Multiple DNA sequences on chromosomes have been detected simultaneously using the fluorescence in situ hybridization approach disclosed in P. M. Nederlof, et al. (1990),
Cytometry
11, 126-131. In this method, alphoid and satellite DNA probes specific for human chromosomes were used to detect chromosomal aberrations. T. Ried, et al. (1992),
Proc. Natl. Acad. Sci. USA
89, 1388-1392, expanded the fluorescence in situ hybridization technique through use of an epifluorescence microscope equipped with a digital imaging camera and computer software that allowed pseudocoloring and image merging. With this system it was possible to distinguish seven different probes using three fluorochromes. Although probe-based pathogen diagnosis is mentioned in Ried et al., the probes disclosed are limited to chromosome-specific alphoid DNAs and to probes containing sequences mapped to particular chromosomes. The probes of Ried et al. were capable of distinguishing different human chromosomes or of distinguishing the locus of a particular sequence on a single chromosome. Dirks et al. (1991)
Exp. Cell Research
194, 310-315, discloses use of fluorochrome-labeled oligonucleotides in combination with oligonucleotides labeled with biotin-11-dUTP and digoxigenin-11dUTP for in situ hybridization studies of three closely related mRNAs.
SUMMARY OF THE INVENTION
The present inventors have discovered a method of assaying for multiple nucleic acid sequence analytes in a single fluid sample. The method of the invention distinguishes different nucleic acid sequence analytes present in a biological sample through use of a plurality of species of capture probes, each species comprising a bacteriophage linked to an oligonucleotide complementary to one analyte. Each bacteriophage is capable of generating a distinctive signal when plated on an indicator medium. In a preferred embodiment, the oligonucleotide moiety of each capture probe species contains a sufficient number of contiguous ribonucleotides to provide a DNA/RNA or RNA/RNA substrate when hybridization to nucleic acid sequence analytes occurs. In this embodiment, after hybridization to nucleic acid sequence analytes has occurred, the hybridization mixture is treated with a ribonuclease capable of cleaving DNA/RNA or RNA/RNA duplexes. In another embodiment, the oligonucleotide moieties of each capture probe species contains only deoxyribonucleic acids, and the hybridization mixture is treated with an appropriate restriction endonuclease to cleave DNA/DNA hybrids formed. Treatment of the hybrids with the ribonuclease or restriction endonuclease releases infective bacteriophage. Each species of capture probe may be detected, and optionally quantitated, by adding the bacteriophage to a bacterial host strain, allowing infection to proceed, and detecting the distinctive signals in bacteriophage plaques produced on an indicator medium. In this way multiple nucleic acid sequence analytes in a single biological sample may be detected.
In one embodiment, the invention provides an assay to detect at least two different nucleic acid sequence analytes in a fluid, comprising the steps of: processing the fluid to release single-stranded nucleic acids; providing at least two species of capture probes, each species comprising a bacteriophage linked to a single-stranded oligonucleotide complementary to one analyte, wherein the bacteriophage of each species is capable of generating a distinctive signal when plated on an indicator medium; combining the fluid and capture probes at a temperature and for a time sufficient to form a hybridization mixture without irreversibly destroying the infectivity of the bacteriophage; incubating the hybridization mixture with a nuclease capable of cleaving nucleic acid duplexes formed in the hybridization step, at a temperature and for a time sufficient to allow cleavage of said duplexes, thereby releasing infective bacteriophage; plating the infective bacteriophage on the indicator medium; and detecting the signals corresponding to the infective bacteriophage.
In another embodiment, the invention provides a kit comprising at least two species of capture probes, each species comprising a bacteriophage linked to a single-stranded oligonucleotide complementary to one analyte, wherein the bacteriophage of each species is capable of generating a distinctive signal when plated on an indicator medium.


REFERENCES:
patent: 5679510 (1997-10-01), Ray et al.

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