Multiplexed detection of biological agents
First Claim
1. A method for detecting at least first and second biological agents, the first agent comprising first and second markers, and the second agent comprising third and fourth markers, the method comprising:
- (a) preparing first and second mixtures from at least one sample suspected of containing the agents;
(b) detecting the presence or absence of markers consisting of the first and third markers in the first mixture in a first container; and
(c) detecting the presence or absence of markers consisting of the second and fourth markers in the second mixture in a second container;
whereby the presence of the first and second markers indicates the presence of the first biological agent in the sample, and the presence of the third and fourth markers indicates the presence of the second biological agent in the sample.
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Accused Products
Abstract
Described are methods, kits and systems for multiplexed detection of biological agents in a sample, e.g., multiplexed detection of e.g., bacteria, viruses, and biological toxins. The method utilizes two markers for each agent; the presence or absence in a sample of each of the two markers per agent is determined in separate reactions; however each reaction is used to detect a single marker for multiple agents. Also disclosed is the multiplexed detection method using real time PCR. The invention provides an efficient, cost-effective, and specific method for multiplexed detection of biological agents.
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Citations
35 Claims
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1. A method for detecting at least first and second biological agents, the first agent comprising first and second markers, and the second agent comprising third and fourth markers, the method comprising:
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(a) preparing first and second mixtures from at least one sample suspected of containing the agents; (b) detecting the presence or absence of markers consisting of the first and third markers in the first mixture in a first container; and (c) detecting the presence or absence of markers consisting of the second and fourth markers in the second mixture in a second container; whereby the presence of the first and second markers indicates the presence of the first biological agent in the sample, and the presence of the third and fourth markers indicates the presence of the second biological agent in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for optically detecting the presence or absence of a number of biological agents greater than the number of color channels used to detect the presence or absence of the biological agents, each of the biological agents having respective first and second nucleic acid sequences that differentiate the biological agent from the other biological agents, the method comprising the steps of:
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a) forming first and second mixtures in first and second containers, respectively, from at least one sample suspected of containing the biological agents, wherein; i) the first mixture comprises reagents and probes, wherein said probes consist essentially of, for each of the biological agents, a respective first probe set for labeling the first nucleic acid sequence of the biological agent; ii) the second mixture comprises reagents and probes, wherein said probes consist essentially of, for each of the biological agents, a respective second probe set for labeling the second nucleic acid sequence of the biological agent; iii) at least two of the first probe sets in the first mixture have the same emission wavelength ranges to be detected in the same color channel, and the at least two corresponding second probe sets in the second mixture have different emission wavelength ranges to be detected in different color channels; b) optically reading the presence or absence of probe signals from the at least two of the first probe sets in the first mixture have the same emission wavelength ranges; c) optically reading the presence or absence of probe signals from the at least two corresponding second probe sets in the second mixture have different emission wavelength ranges; and d) determining from the combination of probe signals received from each of the mixtures the presence or absence of the biological agents.
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16. A method for detecting at least first and second biological agents, the first agent comprising first and second markers, and the second agent comprising third and fourth markers, the method comprising:
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a) preparing a first mixture in a first container from at least one sample suspected of containing the agents; b) detecting the presence or absence of markers consisting of the first and third markers in the first container; c) if either of the first or third markers is present in the first container, preparing a second mixture in a second container from the at least one sample; and d) detecting the presence or absence of markers consisting of the second and fourth markers in the second container; whereby the presence of the first and second markers indicates the presence of the first biological agent in the sample, and the presence of the third and fourth markers indicates the presence of the second biological agent in the sample. - View Dependent Claims (17, 18)
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- 19. A kit for detecting at least first and second biological agents, the first agent comprising first and second markers, and the second agent comprising third and fourth markers, the kit comprising at least first and second containers, wherein the first container houses reagents and a first probe set consisting essentially of a first probe specifically recognizing the first marker and a second probe specifically recognizing the third marker, and wherein the second container houses reagents and a second probe set consisting essentially of a third probe specifically recognizing the second marker and a fourth probe specifically recognizing the fourth marker.
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29. A system for detecting at least first and second biological agents, wherein the first agent comprises first and second markers, and the second agent comprises third and fourth markers, the system comprising:
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(a) at least first and second containers, the first container housing reagents and probes for detecting markers consisting of the first and third markers and the second container housing reagents and probes for detecting markers consisting of the second and fourth markers; (b) at least one detector arranged to detect the presence or absence of the markers in the containers; and (c) at least one controller in communication with the at least one detector, the controller being programmed with computer-readable instructions to perform a series of operations comprising; i) initiating a detection reaction in the first container; ii) receiving data from the detector; and iii) determining from the data the presence or absence of the first and third markers in the first container; wherein if the first or third markers is present in the first container, the controller performs a second series of operations comprising; iv) initiating a second detection reaction in the second container; v) receiving additional data from the detector; and vi) determining from the additional data the presence or absence of the second or fourth markers in the second container; whereby the presence of the first and second markers indicates the presence of the first biological agent in a sample, and the presence of the third and fourth markers indicates the presence of the second biological agent in the sample. - View Dependent Claims (30, 31, 32, 33)
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34. An automated system for determining the presence or absence of a plurality of agents, each of the agents comprising respective first and second nucleic acid sequences that differentiate the agent from the other agents, the automated system comprising:
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a) at least one temperature control system for subjecting first and second reaction mixtures suspected of containing the agents to nucleic acid amplification conditions, the first reaction mixture consisting essentially of reagents and probes for amplifying and detecting the first nucleic acid sequence of each of the agents, and the second reaction mixture consisting essentially of reagents and probes for amplifying and detecting the second nucleic acid sequence of each of the agents; b) at least one detection mechanism arranged to detect probe signals from the reaction mixtures; and c) at least one controller in communication with the at least one temperature control system and with the at least one detection mechanism, the controller being programmed to perform the steps of; i) sending control signals to the temperature control system to subject the first reaction mixture to nucleic acid amplification conditions; ii) determining from probe signals received from the first reaction mixture if the first nucleic acid sequence of any of the agents is present in the first reaction mixture; iii) if the first nucleic acid sequence of any of the agents is present in the first reaction mixture, then sending control signals to the temperature control system to subject the second reaction mixture to nucleic acid amplification conditions; and iv) determining from probe signals received from the second reaction mixture if the second nucleic acid sequence of any of the agents is present in the second reaction mixture; whereby the presence of the first and the second nucleic acid sequences of any of the agents is indicative of the presence of that agent. - View Dependent Claims (35)
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Specification