WO2005009202A2 - Automatic identification of bioagents - Google Patents

Abstract

Automated rapid identification of unknown bioagents is enabled using a self-contained device coupled to a system executive module. The device includes two decks separated by an airlock and a storage area for storing reagents and filter plates for use in identifying bioagents. A programmable robot assembly isolates and purifies DNA from the bioagent in the first deck and prepares the sample for PCR. The first deck is preferably maintained at a higher atmospheric pressure than the area external to the device, while the second deck is maintained at a lower atmospheric pressure than the external area to avoid contamination. After isolation and purification, the sample is passed through the airlock to the second deck, where it undergoes PCR. After washing and eluting of the sample, the molecular mass of the sample is determined by mass spectroscopy. The system executive provides automated request handling, job scheduling, resource management and drill-down analysis.

Description

AUTOMATIC IDENTIFICATION OF BIOAGENTS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application Number 60/470.175, filed on May 12, 2003, which is incorporated by reference herein in its entirety.

[0002] This application is also related to United States Patent Application Publication 20030082539 Al, serial no. 09/891,793, entitled "Secondary Structure Defining Database And Methods For Determining Identity And Geographic Origin Of An Unknown Bioagent Thereby," filed on Jun 26, 2001, and incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

[0003] The present invention relates generally to the field of investigational bioinf ormatics and more particularly to devices and methods for identifying bioagents.

Description of the Related Art

[0004] In the United States, hospitals report well over 5 million cases of recognized infectious disease-related illnesses annually. Significantly greater numbers remain undetected, both in the inpatient and community settings, resulting in substantial morbidity and mortality. Critical intervention for infectious disease relies on rapid, sensitive and specific detection of the offending pathogen, and is central to the mission of microbiology laboratories at medical centers. Unfortunately, despite the recognition that outcomes from infectious illnesses are directly associated with time-to-pathogen recognition, as well as accurate identification of the class and spedes of microbe and ability to identify the presence of drug resistance isolates, conventional hospital laboratories often remain encumbered by traditional slow multi-step culture-based assays. Other limitations of the conventional laboratory include: extremely prolonged wait-times for pathogens with long generation time (up to several weeks); requirements for additional testing and wait times for speciation and identification of antimicrobial resistance; diminished test sensitivity for patients who have received antibiotics; and absolute inability to culture certain pathogens in disease states associated with microbial infection.

[0005] Rapid and definitive microbial identification is desirable for a variety of industrial, medical, environmental, quality, and research reasons. Traditionally, the microbiology laboratory has functioned to identify the etiologic agents of infectious diseases through direct examination and culture of specimens. Since the mid-1980s, researchers have repeatedly demonstrated the practical utility of molecular biology techniques, many of which form the basis of clinical diagnostic assays. Some of these techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and separation and purification of nucleic acids (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). These procedures, in general, are time-consuming and tedious. Another option is the polymerase chain reaction (PCR) or other amplification procedure which amplifies a specific target DNA sequence based on the flanking primers used. Detection and data analysis then convert the hybridization event into an analytical result.

[0006] Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. Low-resolution MS may be unreliable when used to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism.

[0007] Antibodies face more severe diversity limitations than do arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.

[0008] Co-owned US Patent Application Publication 20030082539 Al describes a method of identifying an unknown bioagent using a database of known bioagents. The nucleic acid from the unknown bioagent is first contacted with at least one pair of "intelligent" oligonucleotide primers that hybridize to sequences of the nucleic acid that flank a variable nucleic acid sequence of the bioagent. Using PCR technology, an amplification product of this variable nucleic acid sequence is made. After standard isolation, the molecular mass of this amplification product is determined using mass spectroscopy techniques. This molecular mass is compared to the molecular mass of known bioagents within the database to identify the unknown bioagent.

[0009] Each of these steps is conventionally performed in a laboratory using a wide array of separate equipment. At each step, the sample is manually transported from one piece of equipment to the next, and an operator manually controls the process. Great care must also be taken to avoid contamination of the sample prior to PCR, as even trace amounts of amplicon can destroy a sample's usefulness. [0010] What is needed is a system and method for providing detection and identification of bioagents using an automated, self-contained device that includes logic for operating independently of operator input, that provides protection from sample contamination, and that can automatically perform drill-down analysis on a sample to further specify the sample's identity.

SUMMARY OF THE INVENTION

[0011] The present invention enables automated rapid identification of unknown bioagents using a self-contained device coupled to a system executive module. The device preferably includes two decks separated by an airlock, and in addition includes a storage area for storing reagents and filter plates for use in identifying bioagents. A programmable robot assembly isolates and purifies DNA from the bioagent in the first deck, preparing the sample for amplification through a polymerase chain reaction (PCR). The first deck is preferably maintained at a higher atmospheric pressure than the area external to the device, while the second deck is maintained at a lower atmospheric pressure than the external area; the pressure differential avoids contamination of the sample prior to amplification. After isolation and purification of the sample, it is passed through the airlock to the second deck, where it undergoes the PCR process. Preferably, PCR can be carried out using several PCR plates, and samples can be transferred to, for example, a 384-well tray. After washing and eluting of the sample in the second deck, the molecular mass of the sample is determined by mass spectroscopy. The system is preferably designed so that the second deck may operate without the first deck in the case where the laboratory has an alternative procedure for isolating and purifying DNA from the bioagent, and preparing the sample for amplification through a polymerase chain reaction (PCR).

[0012] The present invention also includes a system executive for controlling operation of the self-contained device. The system executive provides a user interface (UI) for receiving analysis requests, and includes logic for performing resource management, job scheduling and additional drill-down analysis. The system executive is also in communication with a database of catalogued bioagents, which in one embodiment forms part of the device, and in alternative embodiments is located remotely. Using the database of bioagents and their known masses, the system executive either identifies the exact bioagent (or its nearest known relative, if it is a new organism not in the database) using the amplicon mass determined through mass spectroscopy (MS), or alternatively determines that a drill-down analysis is required to further differentiate the sample from among multiple candidates within a related group. In the latter case, the system executive automatically chooses an appropriate set of amplification primers and instructs the device to repeat the PCR process with the second set of primers, obtaining a second identification through MS. This second amplicon mass is then matched once again against catalogued bioagents in the database, and the previously unresolved bioagent sample is identified with a higher degree of precision. Because the system can function autonomously for extended periods, and because of its small footprint and the ability to swap in and out its modular components, for example as they are upgraded or repaired, the present invention makes bioagent identification more accessible to treatment and detection centers, enabling a more rapid response than is attainable using conventional technology. In addition, the present invention includes alerting capability for automatically notifying authorities that a bioagent associated with a high threat level has been identified.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig. 1 is a block diagram illustrating basic components of a device in accordance with an embodiment of the present invention.

[0014] Fig. 2 is a block diagram illustrating a system executive in accordance with an embodiment of the present invention. [0015] Fig. 3 is a flowchart illustrating a method for identifying a bioagent in accordance with an embodiment of the present invention.

[0016] Fig.4 is a block diagram illustrating a layout of a device in accordance with an embodiment of the present invention.

[0017] Fig.5 is a flowchart illustrating a method for performing DNA isolation in accordance with an embodiment of the present invention.

[0018] Fig. 6 is a flowchart illustrating a method for performing PCR preparation in accordance with an embodiment of the present invention.

[0019] Fig. 7 is a flowchart illustrating a method for archiving a sample in accordance with an embodiment of the present invention.

[0020] Fig. 8 is a flowchart illustrating a method for performing PCR in accordance with an embodiment of the present invention.

[0021] Fig. 9 is a flowchart illustrating a method for performing plate transfers in accordance with an embodiment of the present invention.

[0022] Fig. 10 is a flowchart illustrating a method for performing an initial rinse in accordance with an embodiment of the present invention.

[0023] Fig. 11 is a flowchart illustrating a method for performing a NH₄HC0₃ wash in accordance with an embodiment of the present invention.

[0024] Fig. 12 is a flowchart illustrating a method for performing a 50% methanol wash in accordance with an embodiment of the present invention.

[0025] Fig. 13 is a flowchart illustrating a method for performing production elution in accordance with an embodiment of the present invention.

[0026] Fig. 14 is a flowchart illustrating a method for performing sample injection in accordance with an embodiment of the present invention. [0027] The figures depict preferred embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Fig. 1 illustrates a device 100 for performing bioagent detection in accordance with a preferred embodiment of the present invention. Device 100 includes two decks, Deck A 102 and Deck B 104, separated by an airlock 106. Samples are received from storage area 408 at Deck A, where their DNA is isolated and purified, and the samples are then prepared for PCR, and sent through airlock 106 to Deck B. In Deck B, they are thermocycled, which performs PCR on the sample. Amplified DNA is then purified, and the amplified and purified samples are then injected into a mass spectrometer for identification. Also shown in Fig. 1 is system executive kOO, further described next with reference to Fig. 2. Purified, in this context, means separated from at least one or more components present in the amplified sample.

[0029] It is helpful for understanding the operation of device 100 to first discuss the operation of system executive kOO. Fig. 2 illustrates a block diagram of system executive kOO in accordance with an embodiment of the present invention. System executive kOO provides logic for operation of device 100, preferably from the receiving of an initial analysis request through to providing a response to the request. Typically, this involves receiving a request from a user through a user interface, determining resource requirements and scheduling for satisfying the request, instructing the device 100 to actually perform the required operations, analyzing the results, and reporting the results to the user. In one embodiment, system executive kOO includes a UI 202; an analysis request controller 204; a sample state controller 206; analysis plans database 210; a waiting-for-resources queue 208; a protocol resource allocator 212; a ready queue 214; scheduling controller 216; active queue 218; device controller 220; analysis server 222; resource manager 224; and drill- down PCR logic 226. We describe the functionality of each of these components in turn.

[0030] UI 202 provides an interface for communicating with system executive kOO. UI 202 may be implemented in a variety of ways, and in a preferred embodiment includes a Simple Object Access Protocol (SOAP) interface, which provides for accessing the UI 202 through XML in a platform-independent fashion. In other embodiments, UI 202 can be implemented through well-understood interfaces such as HTML, Java, and the like.

[0031] When a user or application — more generally, a requestor — rovides a request to system executive 100, UI 202 calls analysis request controller (ARC) 204. ARC 204 determines based on requestor input what type of identification is being requested. For example, the requestor may wish to know which family of bacteria a particular sample is suspected to contain. Alternatively, the requestor may want to know the specific strain of a virus suspected to be present in a sample. ARC 204 preferably provides the requestor with a hierarchical view of options for specifying the type of analysis desired, and preferably displays only those options that system 100 can provide depending on available devices, resources and the incoming sample type. ARC 204 passes properly formulated requests to sample state controller 206.

[0032] Sample state controller 206 uses the request received from ARC 204 to determine an appropriate analysis plan. An analysis plan includes a selection of protocols to be performed on the sample in order to obtain an appropriate response to the request. Sample state controller 206 preferably includes a mapping from request types to analysis plans. In a preferred embodiment, analysis plans are stored in an analysis plan database 210, though any suitable data structure can be used, as will be appreciated by those of skill in the art.

[0033] Having determined from the retrieved analysis plan which protocols are to be executed, sample state controller 206 inserts the protocols into waiting-for- resources queue 208. Waiting-for-resources queue 208 is a queue that holds protocols that are to be executed, but have required input resources not yet allocated. Protocols remain in the waiting-for-resources queue 208 until moved to ready queue 214 by protocol resource allocator 212 or cancelled, e.g., by a user request.

[0034] Protocol resource allocator 212 periodically examines the waiting-for- resources queue 208 to see which protocols are awaiting scheduling. A protocol includes a list of resources and associated amounts required by the protocol. Resources are various materials and reagents required for processing and can include, for example, disposable pipette tips, microtiter filter plates, plates of amplified DNA, and data files. The protocol resource allocator determines whether sufficient amounts of required resources are available to allow a protocol to run. As resources required by a protocol become available, they are reserved for the protocol. When a protocol has all required resources reserved for it, the protocol resource allocator 212 moves the protocol to the ready queue 214. Protocol resource allocator 212 knows which resources are available to assign to protocols by communicating with resource manager 224. Resources are marked reserved in the resource manager as they are assigned to specific protocols.

[0035] Protocols are preferably either testing protocols or analysis protocols. A testing protocol specifies operations to be performed on the sample by device 100, e.g., by equipment located on Deck A 102 and Deck B 104. An analysis protocol specifies an analysis to be performed by analysis server 222. While examples of resources in a testing protocol include PCR plates, dispensing tips, etc., a resource in an analysis protocol can be anything that must be supplied to analysis server 222 to perform the analysis, such as a data file including results from mass spectroscopy testing.

[0036] Resource manager 224 maintains an inventory of which resources are available for use in device 100. When a resource is added to device 100, resource manager 224 receives a notification from device controller 220. In one embodiment, resources are identified by bar codes, and resource manager 224 includes logic to determine a particular resource type associated with a particular bar code received from device controller 220. In one embodiment, when a resource is added to device 100, the notification received from device controller 220 is the bar code of the added resource. Upon receiving the notification, resource manager 224 updates its inventory list to include the newly added resource. When a resource is reserved for a specific protocol, resource manager 224 decrements its inventory list to exclude the allocated resource.

[0037] Ready queue 214 includes protocols that have been assigned specific resources and are waiting to run. Scheduling controller 216 periodically examines the ready queue 214 for protocols, and when it finds a protocol sends it to either device controller 220 in the case of a testing protocol, or to analysis server 222 in the case of an analysis protocol. Scheduling controller 216 also preferably puts a copy of the protocol in active queue 218 so that system executive kOO maintains a real-time record of which protocols are currently being run.

[0038] Device controller 220 receives a testing protocol from scheduling controller 216 and performs the operations specified in the protocol as described further below. Device controller 220 reports the results of the operations to schedule controller 216. Typically, an analysis protocol in waiting-for-resources queue 208 will be run upon indication that a testing protocol has been completed (for example, because the test data produced by the test protocol is a resource for the analysis protocol). Analysis server 222 reports the results of the analysis to scheduling controller 216, which in turn reports back to sample state controller 206. Sample state 206 then either returns a response to the requestor via UI 202, or else determines according to drill-down logic 226 that additional analysis is required. Methods by which analysis server 222 identifies a bioagent from BCS data are further described in co-owned US Application 20030082539 Al.

[0039] As noted, a requestor is preferably able to specify a type of identification to be performed on the sample. If the type of identification is of a more general nature, such as identifying a bacterial family to which the sample belongs, the initial set of protocols using intelligent primers may be sufficient to provide a response to the request. However, where a more specific identification has been requested, and, for example, different strains of an organism have very similar or identical base composition sequences, it is useful to perform an additional drill-down analysis by performing a second set of PCR reactions using more focused primers followed by an additional round of mass spectroscopy. For example, Bacillus anthracis can be distinguished from Bacillus cereus and Badllus thuringiensis using the primer 16S_{971 1062}. Additional primer pairs that produce unique base composition signatures are described in co-owned US Patent Application 20030082539 Al.

[0040] When sample state controller 206 receives the results of the initial analysis from analysis server 222 (via scheduling controller 216), it compares the results obtained with the request received from the requestor. If the results are sufficient to respond to the request, then the response is forwarded through UI 202 to the requestor. If the results are not spedfic enough to respond to the request, sample state controller 206 uses drill-down logic 226 to automatically select an appropriate pair of primers for performing additional PCR. Protocols for performing the additional PCR, associated mass spectroscopy, and analysis are determined and forwarded to waiting-for-resources queue 208. The protocols are run as described above, results are received from analysis server 222, and once again evaluated by sample state controller 206. As will be appreciated by those of skill in the art, the drill-down process can be automatically repeated if needed to provide even more focused results.

[0041] Fig. 3 provides a flowchart illustrating a method of operation of system executive kOO in one embodiment of the present invention. System executive kOO receives 302 a request from a requestor and selects 304 an appropriate analysis plan from its store 210 of analysis plans based on the request. Protocols associated with the analysis plans are placed 306 into a waiting-for-resources queue. Protocol resource aUocator 212 inspects the waiting-for-resources queue 208 and allocates 308 resources to protocols in the queue as the resources become available. Once all resources required for a process have been aUocated, the protocol is moved 310 from waiting-for-resources queue 208 to ready queue 214. If 312 the protocol is a test protocol, scheduling controller 216 sends 314 the protocol to device controller 220 for execution, and receives 316 the results back from device controller 220 upon termination. Alternatively, if 312 the protocol is an analysis protocol, then the protocol is sent 318 to analysis server 222 for analysis, and the results of the analysis are received 320 back from the server 222 upon completion of the analysis. Results received either from device controller 220 or analysis server 222 are then forwarded 322 to sample state controller 206. If 324 the results satisfy the request, then system kOO reports 326 the results to the requestor. If 324 the results do not satisfy the request, then drill-down PCR logic 226 is used to select 328 new primers, and appropriate protocols are placed 306 once again in the waiting-for-resources queue 208 so that drUl-down PCR analysis can be completed.

[0042] As noted, device controller 220 receives testing protocols to be run from scheduling controller 216. Device controller 220 executes the testing protocols by operating the components of device 100 to perform the functions described in the protocols. Fig. 4 provides a more detailed view of the surface of device 100 in one embodiment. Those of skill in the art will appreciate that the particular arrangement Ulustrated in Fig. 4 is not the only arrangement possible, and the actual placing of components can be changed without departing from the spirit of the present invention.

[0043] Fig. 4 indudes Deck A 102, Deck B 104, and storage area 110. Deck A 102 includes a storage robot 402 for deUvering resources to Deck A 102 from storage 110 and returning the resources to storage 110 on demand. Note that as ulustrated, resources always make their way through Deck A 102 even if going to Deck B 104, and resources preferably never return to storage from Deck B 104. This arrangements prevents contamination of anything in Deck A 102, where un-amplified samples reside, with amplified DNA from Deck B 104. Deck A 102 also includes a conveyor 404 for transporting objects; a gantry pickup point 406, which is an area of Deck A 102 accessible to a gantry of robot 408; robot 408 which is responsible for implementing the various testing protocols by moving resources and samples from one location to another, and for operating the testing components; and a heat sealer 410 for sealing plates. Also shown is airlock 108, which separates storage area 110 from Deck A 102.

[0044] Deck B 104 includes a plate shaker (not shown); a centrifuge 414; thermocyclers 416; a 96-channel pipette 418; a heat sealer 420; a manifold liquid dispenser 422; an autosampler 424; and mass spectrometers 426. Also shown is airlock 106, which separates Deck A 102 and Deck B 104, aUowing different atmospheric pressures to exist on each deck. The operation of the various components of device 100 is now described further.

[0045] Referring now to Fig. 5, there is shown a flow chart illustrating the operation of device 100 and its constituent components as it carries out DNA isolation in accordance with an embodiment of the present invention. To begin, an initial setup function is performed by an operator prior to use of the device, which indudes stocking 502 the device with an initial inventory of resources. After this initial resource setup, operator intervention is preferably required only to replenish resources that have been consumed, or to add new samples to be processed. In one embodiment, the initial setup includes loading of various reagents, tips for transferring Uquid between plates, filling of the reagent storage reservoir with elution buffer, and washing the buffers.

[0046] After the initial setup operation, or, alternatively, verification by the device controller 220 that sufficient resources are already avaUable, DNA isolation can begin. A sample containing the bioagent to be identified is located on a sample source plate. The sample source plate is placed 504 on conveyer belt 404 by storage robot 402, and passed from storage location 408 to Deck A 102 through airlock 108 for transport 506 to gantry pickup point 406. Note that gantry pickup point 406 is a location from which the robotic handler that moves materials between stations on Deck A can retrieve an object. At this point, a filter plate is presented 508 by storage robot 402. Meanwhile, the sample source plate has preferably reached gantry pickup point 406 and is now transported 510 to the robot 408, and the filter plate now reaches gantry pickup point 406. An elution plate is then presented 516 by storage robot 402 to conveyer 406, just as the filter plate reaches 518 robot 408. Conveyer 406 transports 520 the elution plate to gantry pickup location 406. The gantry then deUvers 522 the elution plate to robot 408. Next, a DNA isolation procedure occurs 524 at robot 408. In a preferred embodiment, this isolation indudes four steps. First, a ceU lysis buffer is added to the sample source plate. Next, the samples are transferred to the filter plate, where the filter media binds the sample. Third, impurities are washed off of the filter using wash buffers, and lastly elution buffer is added to the plate. Purified genomic DNA then elutes onto the elution plate, which is now referred to as a genomic DNA plate.

[0047] After the isolation of DNA, the tip racks are preferably discarded for later removed 526 by an operator. The sample source plate is then moved 528 to and sealed by heat sealer 410. The sample source plate then is moved 530 to a conveyer for transport to a disposal area on Deck B 104. Next, the filter plate is moved 532 to the heat sealer 410 and sealed. Meanwh e, the sample source plate travels 534 to the airlock 106 to Deck B 104 for disposal. The filter plate also travels 536 through airlock 106 to Deck B 104 for disposal.

[0048] Referring now to Fig. 6, there is shown a flow chart illustrating a process for preparing samples for PCR in accordance with an embodiment of the present invention. To begin, storage robot 402 presents 602 a tip box to the Deck A conveyer 404, which carries 604 the tip box to gantry pickup position 406. Primer plate 1 is presented 606 by storage robot 402 to the conveyer 404, as the pipet tip box is moved 608 to robot 408. Primer plate 1 is then deUvered 610 to plate pickup position 406, and then moved 612 to robot 408 by the gantry. PCR setup then begins 614. In a preferred embodiment, PCR setup 614 includes the foUowing steps: first, the sample is aspirated from the first weU of the Genomic DNA plate. Next, the sample is transferred into 16 wells of primer plate 1. This process repeats until primer plate 1 is full. The primer plate is now known as pre-PCR plate 1.

[0049] Pre-PCR plate 1 is moved 616 to sealer 410, which applies 618 a foil seal to pre-PCR plate 1. Next, the gantry moves 620 the tip box back to the conveyer destined for the Deck B waste area. The sealed pre-PCR plate is then moved 622 to conveyer 404 and also headed toward Deck B 104. A new tip box is presented 624 by storage robot 402, and the above steps 604 to 622 are preferably then repeated 626 for each remaining plate.

[0050] Following preparation of the pre-PCR plates, and referring now to Fig. 7, the genomic DNA plate is re-lided 702 by robot 408, placed 704 on the conveyer 404, and returned 706 to the storage robot 402, which places 708 the genomic DNA plate into storage area 110.

[0051] As we shift our attention now to Deck B 104, note again that atmospheric pressure is regulated on Deck A and Deck B in order to avoid contamination of the sample prior to amplification. By keeping pressure lower in Deck B 104 than in Deck A 102, contaminants do not travel back into Deck A from Deck B. It is also valuable to keep the pressure of Deck B 104 at a lower pressure than the ambient room pressure. This differential prevents contaminants (particularly amplified DNA) from escaping from Deck B 104 into the open, which might then aUow some to enter and contaminate Deck A 102.

[0052] Referring now to Fig. 8, there is shown a flow chart illustrating a method for thermocycling PCR samples in Deck B of device 100, in accordance with an embodiment of the present invention. Pre-PCR Plate 1 is received 802 through airlock 106 from Deck A 102, after being processed as described above with respect to Fig. 6. Pre-PCR Plate 1 is then transferred 804 to plate shaker 412 and mixed. Next, a PCR Balance plate is transferred 806 to centrifuge 414. As is known by those of skill in the art, a balance plate is a plate that is of equivalent weight to the plate being centrifuged. Pre-PCR Plate 1 is also transferred 808 to centrifuge 414, and spun down 810. Pre-PCR plate 1 is then moved 812 to thermocycler 416, where it preferably undergoes 814 cycling for about 2.5 hours. The PCR Balance plate is then picked up 816 from the centrifuge and returned to storage. Pre-PCR Plates 2-16 are then received in turn through airlock 106 from Deck A, and undergo a thermocycling process similar to pre-PCR Plate 1.

[0053] After the pre-PCR plates have undergone thermocycling, they are known as crude PCR plates. Referring now to Fig. 9, following thermocycling, the PCR Balance plate is transferred 902 to centrifuge 414. Crude PCR plate 1 is then transferred 904 to centrifuge 414 and spun down 906. Crude PCR plate 1 is then moved 908 to a 96-channel pipette 418, and the PCR Balance plate is returned 910 to storage.

[0054] Next, a resin slurry source plate is transferred 912 to 96-channel pipette 418, and its lid is removed 914. The resin slurry is resuspended 916 by continuous aspirate dispense, and then transferred 918 to crude PCR plate 1. A filter plate, preferably having 384 weUs, is then presented 920 at airlock 106. The resin slurry source plate is reUdded and returned 922 to storage. A binding reaction in crude PCR plate 1 is mixed 924 by continuous aspiration and dispensing, and then transferred 926 to the first well of the filter plate. The needles on 96-channel pipette 418 are then washed 928, the filter plate is moved 930 back to temporary storage, and empty PCR plate 1 is transferred to plate sealer 420 and sealed 932. The sealed empty crude PCR plate 1 is then moved 934 to a solid waste capture area (not shown). The remaining crude PCR plates are then transf erred to the wells of the fUter plate in a similar fashion. The next phase of bioagent detection involves an initial rinse.

[0055] Referring now to Fig. 10, after the last PCR plate has been transferred to the filter plate, the filter plate is moved 1002 to manifold dispense system 422. In a preferred embodiment, about 125 »L of water are dispensed 1004 to each well for balance. The resin capture waste balance is moved 1006 to centrifuge 414, and a waste catchplate arrives 1008 at airlock 106 from Deck A 102 and is also moved 1010 to centrifuge 414. The filter plate is then moved to centrifuge 414 and preferably stacked 1012 on top of the waste catchplate. The filter plate and catch plate assembly is then centrifuged 1014, preferably for about 15 seconds.

[0056] Next, and referring now to Fig. 11, the filter plate is moved 1104 to manifold dispensing system 422 where about 200 »L of NH₄HCO_a is dispensed 1106. The waste catchplate is transferred 1108 to 96-channel pipette 418, where the waste is aspirated off and syringe needles are washed. The empty waste catchplate is then returned 1110 to centrifuge 414. The fUter plate is then returned 1112 to the centrifuge 414, and stacked on top of the waste catchplate. The fUter plate / waste catchplate combination is then spun 1114 on the centrifuge for about 15 seconds. The process is preferably repeated 1116 a second and third time. [0057] Referring now to Fig. 12, following the NH₄HCO₃ the filter plate returns 1202 to the manifold dispensing system 422, where receives about 200 »L of MeOH wash. The waste catchplate is again cleaned 1204, and returned 1206 to centrifuge 414. The filter plate also returns 1208 to centrifuge 414, where the filter plate / waste catchplate combination is again washed 1210 for about 15 seconds. In a preferred embodiment, this cyde is repeated 1212 twice more, with the final washing being for approximately two minutes. The sample is now ready for elution.

[0058] Referring now to Fig. 13, the filter plate returns again 1302 to manifold dispensing system 422, where about 25-50 «L of elution buffer is dispensed. The waste catchplate is again cleaned 1304 and returned 1306 to storage, as is the resin capture waste balance. The resin product capture balance is moved 1308 to centrifuge 414, and a product catchplate is deUvered 1310 to Deck B 104 from Deck A 102 via airlock 106 and also placed in centrifuge 414. Next, the fUter plate is moved 1312 to centrifuge 414 and placed on top of the product catchplate, where the combination is spun 1314 for about 2 minutes. Product is then eluted 1316 from the filter plate to the product catchplate. At this point, the samples are now amplified and purified. The empty fUter plate is then moved to the Deck B sealer 420 and sealed 1318. The sealed fUter fl4 is then moved 1320 to the solid waste capture area. The product catchplate is moved to the Deck B sealer 420 and sealed 1322 to prevent evaporation. The resin capture balance plate is returned 1324 to storage.

[0059] Following elution, and referring now to Fig. 14, the sample is preferably subjected to mass spectroscopy. The sealed product catchplate is moved 1402 to a conveyer and directed to autosampler 424. Samples from the catchplate are sampled 1404 by the autosampler 424 and then sprayed 1406 into mass spectrometer 426. Sampling by autosampler 424 in one embodiment takes approximately 7 hours to complete. The empty product catchplate 424 is then sealed 1408 and moved to solid waste capture fl8. The results of the analysis by mass spectrometer 426 are then made avaUable plO to scheduling controller 216, which proceeds as described above. [0060] As will be appreciated by those of skill in the art, so long as an inventory of resources and samples is maintained, system kOO can operate autonomously. Handling requests, running test protocols, querying the analysis server, determining whether the results are suffident to fulfill the request, and if they -ire not then automatically selecting a secondary set of primers for performing drill-down — each of these processes is automated in system kOO. This automation enables an additional feature of system kOO, in which bioagents have associated threat levels, and threat levels have corresponding reporting actions. In one embodiment, bioagent threat levels are returned by analysis sever 222 along with the bioagent identification. In an alternative embodiment, sample state controller 206 or another module of device 100 includes an assodation between bioagents and threat levels. For example, bioagents associated with terrorist activity may have a corresponding threat level of "very high", while harmless bioagents have a corresponding threat level of "very low". In one embodiment, threat levels differ depending on a geographic origin of the sample, in order to account for bioagents that may be common (and presumably a low threat) in one region, but rare (and thus potentially threatening) in a different region. Sample state controUer 206 preferably includes logic for issuing an alarm, e.g., via UI 202, to appropriate authorities when a bioagent having a threat level above a threshold value is detected.

[0061] The present invention has been described in particular detail with respect to a limited number of embodiments. Those of skill in the art wiU appreciate that the invention may additionally be practiced in other embodiments. First, the particular naming of the components, capitalization of terms, the attributes, data structures, or any other programming or structural aspect is not mandatory or significant, and the mechanisms that implement the invention or its features may have different names, formats, or protocols. Further, the system may be implemented via a combination of hardware and software, as described, or entirely in hardware elements. Also, the particular division of functionality between the various system components described herein is merely exemplary, and not mandatory; functions performed by a single system component may instead be performed by multiple components, and functions performed by multiple components may instead performed by a single component. For example, the particular functions of the sample state controUer 206, scheduling controller 216, and so forth may be provided in many or one module.

[0062] Some portions of the above description present the feature of the present invention in terms of algorithms and symbolic representations of operations on information. These algorithmic descriptions and representations are the means used by those skiUed in the bioi-nf ormatics arts to most effectively convey the substance of their work to others skiUed in the art. These operations, whUe described functionally or logically, are understood to be implemented by computer programs. Furthermore, it has also proven convenient at times, to refer to these arrangements of operations as modules or code devices, without loss of generality.

[0063] It should be borne in mind, however, that all of these and similar terms are to be assodated with the appropriate physical quantities and are merely convenient labels applied to these quantities. Unless spedfically stated otherwise as apparent from the present discussion, it is appredated that throughout the description, discussions utilizing terms such as "processing" or "computing" or "calculating" or "determining" or "displaying" or the Uke, refer to the action and processes of a computer system, or simUar electronic computing device, that manipulates and transforms data represented as physical (electronic) quantities within the computer system memories or registers or other such information storage, transmission or display devices.

[0064] Certain aspects of the present invention indude process steps and instructions described herein in the form of an algorithm. It should be noted that the process steps and instructions of the present invention could be embodied in software, firmware or hardware, and when embodied in software, could be downloaded to reside on and be operated from different platforms used by real time network operating systems.

[0065] The present invention also relates to an apparatus for performing the operations herein. This apparatus may be specially constructed for the required purposes, or it may comprise a general-purpose computer selectively activated or reconfigured by a computer program stored in the computer. Such a computer program may be stored in a computer readable storage medium, such as, but is not limited to, any type of disk including floppy disks, optical disks, CD-ROMs, magnetic-optical disks, read-only memories (ROMs), random access memories (RAMs), EPROMs, EEPROMs, magnetic or optical cards, application spedfic integrated circuits (ASICs), or any type of media suitable for storing electronic instructions, and each coupled to a computer system bus. Furthermore, the computers referred to in the specification may indude a single processor or may be architectures employing multiple processor designs for increased computing capability.

[0066] The algorithms and displays presented herein are not inherently related to any particular computer or other apparatus. Various general-purpose systems may also be used with programs in accordance with the teachings herein, or it may prove convenient to construct more speciaUzed apparatus to perform the required method steps. The required structure for a variety of these systems wUl appear from the description above. In addition, the present invention is not described with reference to any particular programming language. It is appredated that a variety of programming languages may be used to implement the teachings of the present invention as described herein, and any references to spedfic languages are provided for disclosure of enablement and best mode of the present invention.

[0067] Finally, it should be noted that the language used in the specification has been principally selected for readabiUty and instructional purposes, and may not have been selected to delineate or circumscribe the inventive subject matter. Accordingly, the disdosure of the present invention is intended to be iUustrative, but not limiting, of the scope of the invention.

Claims

CLAIMS 1. A computer-implemented method for identifying a bioagent present in a sample, the method comprising: receiving a request from a requestor to identify a bioagent present in a sample; determining from the received request at least one testing protocol to run; instructing a testing device to run the determined protocol; receiving a result from the testing device, the result including a mass of an ampUfication product prepared by the device; comparing the mass of the ampUfication product to expected masses of a set of bioagents; identifying the bioagent based on results of the comparison; and returning to the requestor the identification of the bioagent.

2. The computer-implemented method of daim 1 wherein the ampUfication product is prepared by a polymerase chain reaction.

3. The computer-implemented method of daim 1 wherein the mass of the ampUfication product is determined using mass spectroscopy.

4. The computer-implemented method of daim 1 wherein the request indudes a bioagent type and determining the at least one testing protocol to run further comprises: selecting an analysis plan, the analysis plan associated with the bioagent type and including at least one testing protocol.

5. The computer-implemented method of daim 1 wherein instructing the testing device to run the determined protocol further comprises: determining at least one resource required by the protocol; determining for each resource required by the protocol an amount of the resource required; and allocating the required amount of each required resource to the protocol.

6. A computer program product for identifying a bioagent present in a sample, the computer program product stored on a computer-readable medium and induding instructions configured to cause a processor to execute the steps of: receiving a request from a requestor to identify a bioagent present in a sample; determining from the received request at least one testing protocol to run; instructing a testing device to run the determined protocol; receiving a result from the testing device, the result including a mass of an ampUfication product prepared by the device; comparing the mass of the ampUfication product to expected masses of a set of bioagents; identifying the bioagent based on results of the comparison; and returning to the requestor the identification of the bioagent.

7. The computer program product of claim 6 wherein the amplification product is prepared by a polymerase chain reaction.

8. The computer program product of daim 6 wherein the mass of the ampUfication product is determined using mass spectroscopy.

9. The computer program product of claim 6 wherein the request indudes a bioagent type and determining the at least one testing protocol to run further comprises: selecting an analysis plan, the analysis plan assodated with the bioagent type and including at least one testing protocol.

10. The computer program product of claim 6 wherein instructing the testing device to run the determined protocol further comprises: determining at least one resource required by the protocol; determining for each resource required by the protocol an amount of the resource required; and allocating the required amount of each required resource to the protocol.

11. A system for identifying a bioagent present in a sample, the system comprising: a storage area for housing resources and samples; a first deck, the first deck coupled to the storage area by an airlock and induding: means for performing DNA isolation and purification on the sample; a sealer, for seaUng plates; a robotic handler for moving materials; a second deck, the second deck coupled to the first deck by an airlock and induding: a reaction vessel for contacting a nucleic acid molecule from a bioagent with a plurality of primers and reagents to carry out a nudeic acid ampUfication reaction; a thermocycler for receiving the reaction vessel and programmed to produce a nucleic add amplification product from the bioagent; and a mass spectrometer for receiving the ampUfication product and outputting a mass of the amplification product.

12. The system of claim 11 further comprising: a device controller, communicatively coupled to the storage area, the first deck and the second deck, for receiving testing protocols and causing the protocols to be run using the storage area, the first deck and the second deck.

13. The system of claim 12 further comprising a system executive, the system executive communicatively coupled to the device contioUer, for: receiving a request from a requestor to identify a bioagent present in a sample; determining from the received request at least one testing protocol to run; instructing a testing device to run the determined protocol; receiving a result from the testing device, the result including a mass of an ampUfication product prepared by the device; comparing the mass of the ampUfication product to expected masses of a set of bioagents; identifying the bioagent based on results of the comparison; and returning to the requestor the identification of the bioagent.

14. The system of claim 11 wherein the first deck is maintained at a higher atmospheric pressure than the second deck.

15. The system of claim 14 wherein the second deck is maintained at a lower atmospheric pressure than the ambient atmospheric pressure.

16. A computer-implemented method of identifying a bioagent, the method comprising: hybridizing a nucleic acid molecule with a first pair of primers, the first pair of primers flanking a first nucleic acid region, the mass of which varies among different bioagents; amplifying the first flanked nucleic acid region to produce a first ampUfication product; deteπnining a mass of the first ampUfication product; performing a first comparison between the mass of the first amplification product and expected masses of a set of bioagents, the expected masses stored in a database, to determine a first identification; automaticauy determining a second pair of primers, the second pair of primers determined based on the first identification; hybridizing the nucleic acid molecule with the second pair of primers to form a second flanked nucleic add region; amplifying the second flanked nucleic acid region to produce a second ampUfication product; determining a mass of the second ampUfication product; performing a second comparison between the mass of the second ampUfication product and the expected masses of the set of bioagents; and identifying the bioagent based on a result of the second comparison.