Quantitative analysis; nucleic aicds amplification
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Publication Number
US-8021848-B2
Publication Date
2011-09-20
Expiration Date
Abstract
The invention provides efficient methods for rapidly and sensitively identifying cellular and viral targets in medical, industrial, and environmental samples. The invention labels targets and then detects them using large area imaging. Diagnostic tests based on the invention can be rapid, ultrasensitive, quantitative, multiplexed, and automated. The tests minimize sample preparation and do not require nucleic acid amplification or cell culture. A broad range of cells and viruses can be detected by the tests. Tests based on the invention can deliver the high level sensitivity of nucleic acid amplification tests, the user-friendliness, and speed of immunoassays, as well as the cost effectiveness and quantification offered by microbiological tests. The invention embodies the best attributes of the current diagnostic technologies, while addressing gaps in the diagnostic repertoire.
Core Innovation
The disclosed diagnostic platform provides a method for detecting individual target cells or viruses in a liquid sample, where the targets measure less than 50 microns in at least two orthogonal dimensions. The method uses one or more signaling moieties that specifically bind to the targets and generates a detectable signal by illuminating the individual targets.
The targets are deposited on a detection surface so that they are randomly dispersed in a detection zone at a density of less than 100 target cells per mm2. Individual targets are simultaneously detected by detecting the signal in a section of the detection area using a photoelectric detector, with the longest linear dimension of the section greater than 1 mm and magnification of less than or equal to 5 times.
The method allows one or more signaling moieties that do not bind to the targets to remain in the container prior to detecting, and they are not removed prior to said detecting. The disclosed system architecture further includes category-binding molecules and signaling moieties, with optional selection or capture features to deposit targets onto the detection surface, supporting multiplexing via signal differentiation and/or geometric differentiation.
Claims Coverage
The independent claim set includes one independent claim. The core inventive concept centers on low-density random deposition of small targets and simultaneous, low-magnification photoelectric detection using bound signaling moieties, with an additional claim feature specifying retention of unbound signaling moieties prior to detection.
Detecting individual target cells or viruses with low-magnification photoelectric detection
A method for detecting individual target cells or viruses in a liquid sample, where targets measure less than 50 microns in at least two orthogonal dimensions, including contacting the targets with signaling moieties that specifically bind to the targets, depositing randomly dispersed targets on a detection surface at a density of less than 100 target cells per mm2, illuminating to generate a detectable signal, and simultaneously detecting the individual targets by detecting the signal in a section of the detection area using a photoelectric detector with the longest linear dimension of the section greater than 1 mm and magnification of less than or equal to 5 times.
Non-removal of unbound signaling moieties prior to detection
The method in which one or more signaling moieties that do not bind to the targets are not removed from the container prior to said detecting.
The provided independent claim coverage centers on simultaneously detecting individual, randomly dispersed target cells or viruses using bound signaling moieties and a photoelectric detector over a detection-area section under magnification of less than or equal to 5 times, while leaving unbound signaling moieties in the container prior to detecting.
Stated Advantages
Rapid, sensitive detection and quantification of individual cellular and viral targets in minimally prepared medical, industrial, or environmental samples.
Avoidance of nucleic acid amplification and cell culture.
Inexpensive, user-friendly imaging without microscopy.
Applicability to diverse sample types, including blood, sputum, urine, and respiratory samples.
Enables simultaneous detecting of individual targets in a section of the detection area using a photoelectric detector with magnification of less than or equal to 5 times.
Does not remove signaling moieties that do not bind to the targets prior to detection.
Documented Applications
Imaging and identifying low numbers of bacteria, viruses, and cells using signaling moieties including fluorescence, chemiluminescence, and light scattering.
E. coli immunoassay complex detection using fluorescence object counting for quantitative identification.
Large-area fluorescence imaging of stained Mycobacterium (AFB).
Rapid antimicrobial susceptibility testing using an E. coli tetracycline example with MIC comparable to culture.
Detection of magnetically selected Candida albicans and multiplex or organism scanning including Candida/E. coli multiplex detection.
Virus detection including adenovirus and RSV in solid and liquid phases and enumeration in blood.
Homogenous immunoassay for HIV with internal controls.
Multiplexed scanning using distinct fluorescent channels and nucleic-acid-probe based comprehensive pathogen testing using combinatorial quantum-dot labeling and CCD imaging.
Detection of M. tuberculosis on sputum slides.
Homogenous magnetic/fluorescent immunoassays for E. coli O157:H7.
Live/dead bacterial staining.
Chemiluminescent and film-based detection.
Multiplex pathogen detection including HCV, HIV, HBV, CMV, Candida albicans, Mycoplasma pneumoniae, Legionella pneumophila, Chlamydia trachomatis, Neisseria gonorrhoeae, adenovirus, and influenza A.
Antimicrobial susceptibility testing (MIC).
Lateral flow with high dynamic-range quantification.
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