Patent Application
20110318728
Molecular Diagnostics (MDx) is the fastest growing segment of the In vitro Diagnostic (IVD) market and is projected to be on the order of $10 billion dollars by 2015. These tests are typically more sensitive, more specific and more timely than earlier generation tests and/or provide information that is unavailable with any other approaches. Examples of MDx applications include assessment of a patient's predisposition to diseases, determination of their likely response to therapeutics, and identification of infectious agents either in the standard clinical settings or in the context of bioterrorism or biodefense.
To date, only several hundreds of the approximately 60,000 CLIA (Clinical Laboratory Improvement Amendment) certified laboratories are capable of performing MDx tests. This is a reflection of the complex workflows of current MDx tests and their requirement for highly trained staff in a controlled laboratory environment to execute these tests. The market for MDx is likely to expand outside of the existing CLIA certified laboratories as demands for MDx rises from the emerging retail health clinics and point of care testing markets.
Despite these exploding demands for MDx, the ability to support these tests is declining under the current system. It is estimated that the number of available trained CLIA staff will decrease by 60-70% within the next few years due to the prevailing demographic shift (retirements) and the lack of trained replacement personnel. These trends point to the need to reduce the complexity of these tests to enable their use by more naïve staff, to disconnect them from the traditional clinical labs and make them available in distributed locations closer to the patient.
Diagnostic assays frequently require sample preparation steps in order to remove substances that interfere with the assays and/or to increase the concentration of analytes in the sample. These sample preparation steps for biological samples often include pre-treatment of the sample to disrupt or lyse the cellular materials (for example by mechanical or enzymatic treatments) to release analytes into solution. These solutions, or lysates, are introduced to a reaction area which specifically binds analytes of interest. These reaction areas are then washed to further remove contaminants. Finally, the analytes are eluted from the reaction area for downstream detection by various analytical methods sensitive to the analytes. This “bind, wash, elute” process is a dominant paradigm in sample processing for MDx.
Nucleic acids are a common class of analytes targeted by MDx and must be released from the cellular bodies so that they become accessible for analysis. By way of background and to provide context for the invention,
There are numerous solid phases which can function as a reaction area and which are adapted to bind nucleic acids, some examples are: glass or silica based surfaces (U.S. Pat. Nos. 5,234,809, 6,787,307), carboxylated surfaces (U.S. Pat. No. 5,705,628), or pH sensitive surfaces (U.S. Pat. No. 6,914,137). The lysates are typically combined with a binding buffer prior to their introduction to the solid surface. The binding buffer promotes selective binding of the nucleic acids to the solid phase over other undesirable substances. The exact composition of the binding buffer varies greatly depending on the solid phase that is used, but in the case of silica based materials, a strong chaotrope such as guanidine is usually a major constituent of the binding buffer. Use of chaotrope based binding buffers have advantages in that they can serve as lysis buffer for many sample types, and they also act as a preservative for nucleic acids. Nevertheless, many successful purification processes do not use the binding buffer as the lysis buffer and a dedicated binding buffer must be added to the lysate. Even when a chaotrope based binding/lysis buffer is used, it can be advantageous, as described in U.S. Pat. No. 6,383,393, to add a second binding buffer containing alcohols prior to solid phase binding. Use of a second binding buffer can improve the binding efficiency of nucleic acids to the solid phase but adds an additional fluid transfer step to the overall process.
As
Since their inception, improvements have been made to the solid phase form factor to improve their utility. Current commercially available solid phases typically come in the form of filtration columns or as coated magnetic beads. The QIAGEN line of QlAprep® (Hilden, Germany) products is an example of a spin filter based form factor. Fluids are introduced to the filter by pipetting into the reservoir on top of the filter unit. Centrifugal force is used to drive the fluids through the filter. The lysate and wash are filtered into waste collection tubes, while the elution buffer is filtered into a dedicated collection tube. The Fuji QuickGene®-Mini80 (Tokyo, Japan) is a similar product which uses pressure as the driving force to move fluids through the filter.
An alternative solid phase form factor is the coated magnetic bead. Here the fluids are mixed in a container with the beads and the two are allowed to interact. Fluid exchange is accomplished by placing an external magnet next to the container, attracting the beads to the container wall, removing the old fluid and replacing it with the new fluid of choice. At the end of the process, the eluate is transferred to a separate dedicated collection tube. One potential drawback of the bead based process is that processing of large volumes of lysate requires larger numbers of beads (which can increase cost) and longer times to allow for beads/fluid interaction and subsequent attraction by the external magnet to the wall (which slows the overall process).
When these solid phase form factors were introduced, the fluid transfer and manipulation steps were performed manually. Recently, more automated and integrated systems have been introduced. Examples of more automated systems which employ filter based technologies include the QIAGEN QIACube®, the Fuji QuickGene®-800, and the Cepheid GeneXpert® (Sunnyvale, Calif.). Systems which uses bead based solid phases include the Promega Maxwell®-16 (Madison, Wis.), Roche MagNA Pure® (Indianapolis, Ind.), and Iquum Liat™ (Marlborough, Mass.) Analyzer. All of these systems are able to automate the fluid handling steps of MDx sample preparation to varying levels of success. But this is but the first hurdle to provide MDx access to the insufficiently staffed CLIA labs, the Point of Care (POC) practitioners, or retail health clinics. A compelling set of features important to access these markets include: 1) Simple to use consumable with on-board reagents. Systems with reagent bottles and tubings present challenges to apparatus maintenance and reagent tracking. 2) Inexpensive consumable. 3) Ability to carryout the lysis step in addition to the “bind, wash, elute” sample preparation steps. 4) Effective waste containment for enhanced safety. 5) Flexible enough to employ single or dual binding buffer chemistry. 6) Able to effectively handle large input sample volume. 7) Can process single sample at a time, but configurable to handle multiple samples when needed. This single sample capability is important in the clinical setting for urgent processing of “STAT” samples. 8) Easily adaptable for introduction of the eluate into any downstream consumable for analytical detection. 9) Ease of integration of sample preparation with downstream detection into a single apparatus.
While the existing systems described above may each contain some combinations of these features, none have all of these important features.
An aspect of the disclosure is directed to fluid handling devices. Fluid handling devices comprise: an inlet for receiving a sample; a reagent layer comprising, a substrate having a first surface and an opposing second surface, at least one reagent storage compartment configured to hold a reagent, and a seal in communication with the at least one reagent storage compartment; and a reaction layer having a first surface and an opposing second surface comprising, a reaction area, and an outlet in communication with the reaction area, wherein the reagent layer and reaction layer are adapted and configured to permit movement of at least one of the reagent layer and the reaction layer in a plane relative to the other layer. Moreover, one or more of a reagent layer support extending from the substrate or a reaction layer support can be provided. Additionally, one or more of each of a shoulder, a seal, such as a selectively openable seal, and one or more reagent storage compartments, which may be compressible, may also be provided. The reagent layer may further be configured to comprise two or more reagent storage compartments and further wherein at least two of the reagent storage compartments of the reagent layer are in fluid communication. Where two or more reagent compartments are provided, two or more reagents may be provided as well. The reagent storage compartment may further be configured to comprise a tip. The tip can be configured such that it enables at least one of mixing of material within the reaction area, transfer of material from a first reaction area to a second reaction area, and transfer of material from a reaction area to a fluid collection compartment. Additionally, the tip may further comprises a selectively openable seal. In some configurations, the reaction area may further be configured to comprise one or more filters. Additionally, one or more reaction vessels may be provided, each of which also may include a reagent. In other aspects a fluid collection compartment may be provided. Additional external positioning features may be provided that are adapted and configured to engage the reagent layer with the reaction layer. A fluid collection layer may be provided, which may, for example also be configured to comprise one or more inlets, provide one or more materials adapted and configured to absorb waste, and comprise one or more fluid collection compartments, which can also contain a reagent and can be sealable. Additionally, at least one of the reagent layer and the reaction layer is nestable within the other layer and the reagent compartment of the reagent layer may also be adaptable to form a pressure tight seal in some configurations. The reagent layer and reaction layer can further be adapted and configured to move at least one of about an axis and along and axis. Additionally, at least one of the reagent layer and the reaction layer are adapted and configured to permit movement in a plane relative to the fluid collection layer. In some configurations, the reagent layer and reaction layer are integrally formed. Moreover, one or more external positioning features can be provided that are adapted and configured to engage the reaction layer with the fluid collection layer. The external positioning features can further be adapted and configured to prevent movement of at least one of the reagent layer and the reaction layer relative to the other layer. Additionally, the external positioning features are adapted and configured to at least one of prevent movement of at least one of the reagent layer, the reaction layer and the fluid collection relative to at least one other layer and permit movement of at least one of the reagent layer, the reaction layer and the fluid collection relative to at least one other layer. The reaction layer may also be in fluid communication with a first reagent storage compartment at a first time and a second reagent storage compartment at a second time, e.g. by a channel or a tube or some other mechanism that permits fluid from one storage compartment to access another storage compartment. Any configuration of components or devices can further comprise one or more device identification components. The one or more device identification components are adapted and configured to identify one or more of each of serial number, manufacturer, lot number, date codes, reagent type, reagent volume, reaction area type, process identification, process parameters needed to run the process, and calibration parameter. Additionally, the one or more device identification components may also be associated with at least one or more of the fluid handling device, the reagent layer, the reaction layer, and the fluid collection compartment. Moreover, the one or more device identification components associated with at least one or more of the fluid handling device, the reagent layer, the reaction layer, and the fluid collection compartment are adapted and configured to communicate information between the one or more of the fluid handling device, the reagent layer, the reaction layer, and the fluid collection compartment. The diagnostic device and/or an adapter between the diagnostic device and a fluid handling device can further be adapted and configured to activate the fluid handling device such that the fluid handling device begins processing a sample.
Another aspect of the disclosure is directed to systems adapted and configured to process fluid. Fluid processing systems comprise: a diagnostic device; and a fluid handling device comprising, an inlet for receiving a sample, a reagent layer comprising, a substrate having a first surface and an opposing second surface, at least one reagent storage compartment configured to hold a reagent, a seal in communication with the at least one reagent storage compartment, a reaction layer having a first surface and an opposing second surface comprising, a reaction area, and an outlet in communication with the reaction area, wherein the reagent layer and reaction layer are adapted and configured to permit movement of at least one of the reagent layer and the reaction layer in a plane relative to the other layer. Moreover, one or more of a reagent layer support extending from the substrate or a reaction layer support can be provided. Additionally, one or more of each of a shoulder, a seal, such as a selectively openable seal, and one or more reagent storage compartments, which may be compressible, may also be provided. The reagent layer may further be configured to comprise two or more reagent storage compartments and further wherein at least two of the reagent storage compartments of the reagent layer are in fluid communication. Where two or more reagent compartments are provided, two or more reagents may be provided as well. The reagent storage compartment may further be configured to comprise a tip. The tip can be configured such that it enables at least one of mixing of material within the reaction area, transfer of material from a first reaction area to a second reaction area, and transfer of material from a reaction area to a fluid collection compartment. Additionally, the tip may further comprises a selectively openable seal. In some configurations, the reaction area may further be configured to comprise one or more filters. Additionally, one or more reaction vessels may be provided, each of which also may include a reagent. In other aspects a fluid collection compartment may be provided. Additional external positioning features may be provided that are adapted and configured to engage the reagent layer with the reaction layer. A fluid collection layer may be provided, which may, for example also be configured to comprise one or more inlets, provide one or more materials adapted and configured to absorb waste, and comprise one or more fluid collection compartments, which can also contain a reagent and can be sealable. Additionally, at least one of the reagent layer and the reaction layer is nestable within the other layer and the reagent compartment of the reagent layer may also be adaptable to form a pressure tight seal in some configurations. The reagent layer and reaction layer can further be adapted and configured to move at least one of about an axis and along and axis. Additionally, at least one of the reagent layer and the reaction layer are adapted and configured to permit movement in a plane relative to the fluid collection layer. In some configurations, the reagent layer and reaction layer are integrally formed. Moreover, one or more external positioning features can be provided that are adapted and configured to engage the reaction layer with the fluid collection layer. The external positioning features can further be adapted and configured to prevent movement of at least one of the reagent layer and the reaction layer relative to the other layer. Additionally, the external positioning features are adapted and configured to at least one of prevent movement of at least one of the reagent layer, the reaction layer and the fluid collection relative to at least one other layer and permit movement of at least one of the reagent layer, the reaction layer and the fluid collection relative to at least one other layer. The reaction layer may also be in fluid communication with a first reagent storage compartment at a first time and a second reagent storage compartment at a second time, e.g. by a channel or a tube or some other mechanism that permits fluid from one storage compartment to access another storage compartment. Any configuration of components or devices can further comprise one or more device identification components. The one or more device identification components are adapted and configured to identify one or more of each of serial number, manufacturer, lot number, date codes, reagent type, reagent volume, reaction area type, process identification, process parameters needed to run the process, and calibration parameter. Additionally, the one or more device identification components may also be associated with at least one or more of the fluid handling device, the reagent layer, the reaction layer, and the fluid collection compartment. Moreover, the one or more device identification components associated with at least one or more of the fluid handling device, the reagent layer, the reaction layer, and the fluid collection compartment are adapted and configured to communicate information between the one or more of the fluid handling device, the reagent layer, the reaction layer, and the fluid collection compartment. The diagnostic device and/or an adapter between the diagnostic device and a fluid handling device can further be adapted and configured to activate the fluid handling device such that the fluid handling device begins processing a sample.
Still another aspect of the disclosure is directed to methods for processing a sample. Methods include, for example, obtaining a sample; inserting a sample into a reagent layer further comprising, a substrate having a first surface and an opposing second surface, one or more reagent storage compartments configured to hold a reagent, and one or more seals enclosing the one or more reagent storage compartments, reacting the sample in a reaction layer having a first surface and an opposing second surface comprising, a reaction area, and an outlet in communication with the reaction area, wherein the reagent layer and reaction layer are adapted and configured to permit movement of at least one of the reagent layer and the reaction layer in a plane relative to the other layer; and processing the sample without human interaction with the sample after the step of inserting the sample into the reagent layer. Additional steps of the method can include, for example, one or more of each of the following steps of: delivering at least one processed sample to the diagnostic machine, analyzing the at least one processed sample, one or more of adding a lysis buffer to the sample, adding a binding buffer to the sample, binding the sample to a reaction area, emptying a fluid into a waste container; adding a wash buffer; adding an elution buffer; and eluting the sample, controlling at least one of a temperature, a reaction time, and a motion, analyzing the processed sample, and delivering at least two processed samples to the diagnostic machine. Samples include, biological samples, which includes, but are not limited to, nucleic acids, blood, nasal washes, suspensions of particulates (such as dirt or feces), other cellular suspensions (such as saliva, cheek swabs, scabs, nail clippings, hair, buccal swabs), protein suspensions, mixtures of compounds and the like. Suitable diagnostic devices for use with the method include, for examples, molecular diagnostic devices, polymerase chain reaction devices, isothermal amplification devices, lateral flow devices, devices employing arrays, electrochemical detection devices, optical detection devices, nucleic acid sequencers. The fluid handling device can further be activated such that the fluid handling device begins processing a sample. Activation can be via a network command, an adapter configured to communication between a diagnostic device and the fluid handling device or via the diagnostic device itself.
Yet another aspect of the disclosure is directed to kits for processing a sample. Kits include, for example, a reagent layer comprising, a substrate having a first surface and an opposing second surface, at least one reagent storage compartment configured to contain a reagent, and a seal in communication with the at least one reagent storage compartment; a packaging adapted and configured to house one or more kit components. Another kit could include, for example, a reaction layer comprising a first surface and an opposing second surface, comprising a reaction area, and an outlet in communication with the reaction area; a packaging adapted and configured to house one or more kit components. Additional components of any kit could include one or more of each of filters, a reaction layer having a first surface and an opposing second surface, comprising a reaction area, and an outlet in communication with the reaction area and a fluid collection compartment, reagents, syringes adapted and configured to deliver the reagents to the reagent layer, fluid collection layers, reaction vessels, reaction area columns, eluate collection vessels, adapters to engage a diagnostic device and a fluid handling device or components of a fluid handling device, detectors, device identification components. Suitable reagents include one or more of the following lysis buffers, binding buffers, wash buffers, elution buffers, reaction buffers, dilution buffers, aqueous solutions, organic solutions, protein solutions, and dried reagents.
Additional aspects of the disclosure relate to a communication system. The communication system comprises: a diagnostic device; a fluid handling device comprising an inlet for receiving a sample, a reagent layer comprising a substrate having a first surface and an opposing second surface, at least one reagent storage compartment configured to hold a reagent, and a seal in communication with the at least one reagent storage compartment, and a reaction layer having a first surface and an opposing second surface comprising, a reaction area, and an outlet in communication with the reaction area, wherein the reagent layer and the reaction layer are adapted and configured to permit movement of at least one of the reagent layer and the reaction layer in a plane relative to the other layer; a diagnostic device server computer system; a diagnostic test result module on the server computer system for permitting the transmission of a diagnostic test result from a diagnostic device over a network; at least one of an API engine connected to at least one of the diagnostic device and the fluid handling device to create a message about the diagnostic test result and transmit the message over an API integrated network to a recipient having a predetermined recipient user name, an SMS engine connected to at least one of the diagnostic device and the fluid handling device to create an SMS message about the diagnostic test result and transmit the SMS message over a network to a recipient device having a predetermined diagnostic test result recipient telephone number, and an email engine connected to at least one of the diagnostic device and the fluid handling device to create an email message about the diagnostic test result and transmit the email message over the network to a diagnostic test result recipient email having a predetermined diagnostic test result recipient email address. Additionally, a storing module can be provided on the server computer system for storing the diagnostic test result on the diagnostic device server database. In some configurations, at least one of the diagnostic device and the fluid handling device is connectable to the server computer system over at least one of a mobile phone network and an Internet network, and a browser on the diagnostic test result recipient electronic device is used to retrieve an interface on the server computer system. The system can be configured such that a plurality of email addresses are held in a diagnostic device database (e.g., email addresses of physicians requesting tests, patients for whom tests are performed, law enforcement personnel, etc.) and fewer than all the email addresses are individually selectable from the diagnostic host computer system (e.g., only the email addresses which should receive a particular test result), the email message being transmitted to at least one diagnostic test result recipient email having at least one selected email address. At least one of the diagnostic device and the fluid handling device can also be connectable to the server computer system over the Internet, and a browser on the diagnostic test result recipient electronic device is used to retrieve an interface on the server computer system. Additionally, a plurality of user names are held in the diagnostic device database and fewer than all the user names are individually selectable from the diagnostic host computer system, the message being transmitted to at least one diagnostic test result recipient user name via an API. In other configurations, the diagnostic test result recipient electronic device is connected to the server computer system over a cellular phone network, for example, such that it is in communication with a mobile device. An interface on the server computer system can be provided such that the interface is retrievable by an application on the diagnostic test result recipient mobile device. In that case, the SMS diagnostic test result can then be received by a message application on the diagnostic test result recipient mobile device. Moreover, a plurality of SMS diagnostic test results are received for the diagnostic test result, each by a respective message application on a respective diagnostic test result recipient mobile device. Additionally, at least one SMS engine can be configured to receive an SMS response over the cellular phone SMS network from the diagnostic test result recipient mobile device and stores an SMS response on the server computer system. In some situations, a diagnostic test result recipient phone number ID can be transmitted with the SMS diagnostic test result to the SMS engine and is used by the server computer system to associate the SMS diagnostic test result with the SMS response. The server computer system can also be connectable over a cellular phone network to receive a response from the diagnostic test result recipient mobile device. Additionally, in some configurations, the SMS diagnostic test result includes a URL that is selectable at the diagnostic test result recipient mobile device to respond from the diagnostic test result recipient mobile device to the server computer system, the server computer system utilizing the URL to associate the response with the SMS diagnostic test result. The communications system can also comprise a downloadable application residing on the diagnostic test result recipient mobile device, the downloadable application transmitting the response and a diagnostic test result recipient phone number ID over the cellular phone network to the server computer system, the server computer system utilizing the diagnostic test result recipient phone number ID to associate the response with the SMS diagnostic test result, a transmissions module that transmits the diagnostic test result over a network other than the cellular phone SMS network to a diagnostic test result recipient user computer system, in parallel with the diagnostic test result that is sent over the cellular phone SMS network, and/or a downloadable application residing on the diagnostic test result recipient host computer, the downloadable application transmitting a response and a diagnostic test result recipient phone number ID over the cellular phone network to the server computer system, the server computer system utilizing the diagnostic test result recipient phone number ID to associate the response with the SMS diagnostic test result. The communication system can further be adapted to activate the fluid handling device such that the fluid handling device begins processing a sample.
Another aspect of the disclosure is directed to a networked apparatus or group of apparatuses. The network apparatus comprises: a memory; a processor; a communicator; a display; a fluid handling device comprising an inlet for receiving a sample, a reagent layer comprising a substrate having a first surface and an opposing second surface, at least one reagent storage compartment configured to hold a reagent, and a seal in communication with the at least one reagent storage compartment, and a reaction layer having a first surface and an opposing second surface comprising, a reaction area, and an outlet in communication with the reaction area, wherein the reagent layer and the reaction layer are adapted and configured to permit movement of at least one of the reagent layer and the reaction layer in a plane relative to the other layer. The networked apparatuses can further be adapted to enable activation of the fluid handling device via the network such that the fluid handling device begins processing a sample.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
The fluid handling device 200 can be configured such that a user can add one or more custom reagents as needed. Alternatively, the fluid handling device 200 can be configured to provide one or more reagents on-board while also providing one or more vessels that are accessible and/or customizable by a user to add one or more custom reagents. The fluid handling device 200 may contain one or more reaction vessels 252 to allow automated assembly of any reactions prior to the sample preparation steps. Fluid handling device 200 may also be configurable to contain one or more filters or filter columns 214 as a reaction area to facilitate processing of larger input sample volumes.
Fluid handling device 200 is further configurable to provide one or more of each of on-board waste collection vessel 226 and eluate collection vessel 224. As will be appreciated by those skilled in the art, providing on-board waste collection vessels and eluate collection vessels can further improve safety and reduce sample and processing contamination risks. The eluate collection vessel 224 may further be configured to contain reagents designed to carry out downstream reactions on the purified analytes.
In use, the fluid handling device 200 is actuated by a fluid processing apparatus 260 which contains subcomponents suitable for use in carrying out one or more the fluid processing steps. The fluid handing device and the fluid processing apparatus together teach the fluid handing system. These subcomponents include, but are not limited to one or more of each of, pressure applicator 286, motion controller 288, and temperature controller 292. As shown, fluid processing apparatus 260 processes a fluid handling device 200 which receives an input sample 274 as provided by the user and, subsequent to processing, produces an output sample 276, such as purified nucleic acid (Sample to Processed Sample) wherein the sample had been processed without further human interaction with the sample during at least some of the processing steps shown on
As will be appreciated by those skilled in the art, if a reaction layer is configured to have more than one reaction area elements or columns, then a single sample could be split or separated into two (or more) different columns. For example, if the sample is added into the reaction vessel, then reacted with lysis buffer, the transfer of this material into the column could be split into two columns (or more). In this way you would have more than one output samples. Thus, as would be appreciated, there is no requirement that the columns are the same. One could be optimized to purify DNA and the other could purify proteins.
Still referring to
Examples of commercially available detection apparatus that perform temperature cycled target amplification with optical signal detection are the MiniOpticon™ Real-Time PCR Detection System from Bio-Rad (Hercules, Calif.), the StepOne™ System from Applied Biosystems (Foster City, Calif.), and the Mx3005P® QPCR System from Agilent Technologies (Santa Clara, Calif.). These apparatuses are configured to accept test tube shaped vessels that receive processed samples (purified nucleic acids) and reagents that act to amplify nucleic acid targets and produce a signal that can be detected optically by the apparatus. A Sample to Answer system may be configurable by using the combination of fluid handling device 200, apparatus 260 and one of these commercially available apparatus that performs temperature cycled target amplification with optical signal detection. In this configuration, the elution vessel 224 would simply be the required test tube shaped vessel. Elution vessel 224 would be configurable to contain prepackaged reagents specifically required for temperature cycled target amplification and optical signal generation, for example, buffers, oligonuclotides, nucleotides, enzymes and fluorescent dyes. These reagents could be in a dry state (lyophilized) that become reconstituted upon the introduction of the processed sample by the combination of fluid handling device 200 and apparatus 260. An example of these reagents in a lyophilized state is the illustra PuRe Taq Ready-To-Go™ Beads by GE Healthcare (Waukesha, Wis.).
Another example of an apparatus that perform temperature cycled target amplification with optical signal detection is the 7900HT Fast Real-Time PCR System from Applied Biosystems (Foster City, Calif.). In addition to using test tube shaped reaction vessels, this instrument is also capable of using a Custom TaqMan® Array, a 384 well microfluidic card that allows samples to be run against TaqMan® Gene Expression Assay targets that are preloaded into each of the wells on the card. A Sample-to-Answer system may be configurable by using the combination of fluid handling device 200, apparatus 260 and an apparatus similar to the 7900HT Fast Real-Time PCR System. In this configuration, the elution vessel 224 would simply be a microfluidic card similar to the Custom TaqMan® Array.
Other commercially available detection systems utilize different detection technologies. For example, the TruDiagnosis® Systems from Akonni Biosystems (Frederick, Md.) uses a TruCycler™ Thermal Cycler to amplify the nucleic acid in a TruArray® MicroArray, followed by a TruDx™ Reader to detect the signal. The vessel required for the TruDiagnosis® Systems is the TruArray® MicroArray, which is a microfluidic vessel containing a micro-array of gel-drop biosensors. A Sample-to-Answer system may be configurable by using the combination of fluid handling device 200, apparatus 260 and an apparatus similar to the 7900HT Fast Real-Time PCR System. In this configuration, the elution vessel 224 would simply be a microfluidic card similar to the TruArray® MicroArray.
Another detection technology is the Lateral Flow device exemplified by pregnancy test devices. These devices can be adapted to detect proteins, nucleic acids or compounds. A Sample-to-Answer system may be configurable by using the combination of fluid handling device 200, apparatus 260 and a lateral flow device where the elution vessel 224 is the lateral flow device.
Persons skilled in the art would appreciate that by modifying the form factor of the elution vessel 224, the fluid handling device 200 and the fluid handling apparatus 260 may be coupled with other detection instrumentation. Persons skilled in the art would also appreciate that such an integrated apparatus may be used alone or as part of a larger system as shown in
By using the system described herein, the labor intensive and error prone processes described with respect to
An embodiment of a fluid handling device 300 suitable for use in accordance with the disclosure herein is shown in
The fluid handling device 300 is adapted and configured to fit into and removeably mate with a fluid processing apparatus (see e.g.,
Other suitable seals, such as duck billed valves, or snip-off or tear-off seals to can be used without departing from the scope of the invention. In the case of duck billed valves the entire second surface 313 of reagent layer 310 is coverable with aluminum lidding to improve reagent retention during storage. This lidding is removable by the user or by the fluid processing apparatus prior to use. In the case of tear-off seals, mechanical features on the fluid processing apparatus may open these seals, for example by cutting, twisting, or tearing the seal, prior to compression of the compartment.
A first support 312 forming a male conical support on the first surface 311 of the reagent layer 310 and a female receiving aperture on its second surface 313 can extend from a reagent layer first surface 311 of the planar substrate 302 of the reagent layer 310. The first support 312 is further configurable to enable the reagent layer 310 to freely rotate R about an axis P1 extending out of the plane P2-P3 as further shown in
The reaction layer 320 having a first (upper as depicted in
The fluid collection layer 330 is made up of one or more fluid collection compartments 324, 326. The one or more fluid collection compartments 324, 326 can each have corresponding inlets 332, 328 which face toward the second surface 323 of reaction layer 320. Free rotation, R, of the reaction layer 320 about an axis P1 extending out of the plane P2-P3 as further shown in
The spindle 335 furthermore can be integrated with the fluid handling device 300, integrated with the fluid processing apparatus, or split between the fluid handling device and the fluid processing apparatus. One advantage of having a spindle integrated with the fluid handling device 300 is the unification of the fluid handling device 300 and maintenance of concentricity of reagent layer 310, reaction layer 320, and fluid collection layer 330. Persons skilled in the art would appreciate there are different ways of designing the spindles 335 for fluid processing apparatus and fluid handling device 300 while providing independent motion of reagent layer 310, reaction layer 320 and fluid collection layer 330 without departing from the scope of the invention.
Independent rotations, R, of reagent layer 310 and reaction layer 320 about axis P1 extending out of the plane P2-P3 as further shown in any of
Once a pressure tight seal has been formed between pressure frangible seal 306 and the reaction area 314, compression of reagent compartment 304 will generate sufficient pressure to break seal 306, deliver its content through reaction area 314, where upon the effluent will flow through reaction area outlet 316 into a fluid collection layer 330 (shown in
After evacuation of reagent compartment 304, it may be desirable to break the seal between 306 and reaction area 314 to allow access to a new, previously unused, or partially used reagent compartment.
Reagent layer 310 is separable from reaction layer 320 by application of a pressure from, for example, the spindle 335, associated with the fluid processing apparatus (not shown), into central support 312 (
Reagent layers 310 and reaction layer 320 are independently rotatable of fluid collection layer 330.
It is possible to include other features into the preferred invention to improve the functionality of the device. For example, it may be desirable to include absorbent materials (not shown) in fluid collection compartment 326 to absorb any liquid waste received from reaction area outlet 316. In addition, through independent motions of fluid collection layer 330 and reaction layer 320, reaction area outlet 316 may be raised up and down to touch this absorbent material in order to wick off any liquid that may adhere to reaction area outlet 316. Filter materials (not shown) may be placed around the junctions of reagent layer 310, reaction layer 320 and fluid collection layer 330 to minimize the escape of aerosols, particulates or gases from the fluid handling device. After use, escape of reagents or waste can be minimized and ease of disposal can be maximized by heat or adhesive sealing of layer reagent layer 310, reaction layer 320 and fluid collection layer 330 to each other.
One or more each of the reagent layer 310, reaction layer 320 and fluid collection layer 330 can further be configured to include one or more mechanisms (for example, such as unique device identification 1272 discussed in
In one aspect, bar codes can be used to store limited information about an layer. For example, such bar codes are oftentimes positioned on the layer itself, and configured to include static information (see for example U.S. Pat. No. 6,180,351). However, as will be appreciated by those skilled in the art, while effective at storing certain data, bar codes have some limitations. For example, bar codes are not capable of collecting dynamic information. In fact, if information is to be added after the bar code has been configured, such as information related to the processing, or if information must be changed or erased, the bar code must be replaced by another bar code which has had the new information transposed thereupon. In other words, information contained on bar codes is fixed as of the time the bar codes are made and placed on the layer or device, which is typically at the point of fabrication. Second, the bar codes are limited to the amount of information they can store because of size constraints. For example, unique identifiers which match a layer to its specific layout information, often referred to as “Globally Unique Identifiers” or “GUIDs” or “Universally Unique Identifiers” or “UUIDs”(see for example U.S. Pat. Nos. 5,812,793 and 5,404,523) typically require 128 bit data string. However, a string of such a length when written as a bar code would usually take up about 3 to 4 cm, which is more room than is often available on a substrate adjacent a typical array (which may be less than about 1 cm in any dimension). Thus, oftentimes a second, shorter code is used, where such a shorter code is used to identify the actual unique identifier. However, this technique adds complexity to the array process. Third, a bar code requires the use of a bar code scanner for reading the information contained on the bar code itself. Such ancillary equipment adds to the cost and complexity of data retrieval.
Additionally, a data storage element can be provided in addition to a bar code which is configurable to receive and store large amounts of data. Suitable data storage elements include, for example, magnetic, silicon chip, optical or solid state storage devices (including magnetic or optical disks or tape or RAM, or any other suitable device). In other words, the data storage element is capable of storing a greater amount of data than would be feasible to store on an bar code and will typically have a storage capacity of from one byte to hundreds of bytes of data to multiple tens or even hundreds of megabytes of data or more. As such, typically about 100 bytes to about 500 megabytes of data or more may be stored, usually from about 250 bytes to about 15 megabytes of data may be stored and more usually from about 0.125 megabytes to about 4 megabytes of data may be stored by the data storage element. Data may be stored in the data storage element manually, for example in the case of static data, or automatically, for example in the case of dynamic data. The stored data may be organized into separate or discrete areas. For example, data may be stored in areas that are generally or broadly accessible and/or stored in areas that are secure or protected, i.e., areas that have limited accessibility, e.g., the areas are protected and accessible only if a password is provided, or the like. Furthermore, the data may be stored in a variety of formats, including, but not limited to, raw, processed, encrypted and decrypted formats. In certain embodiments of the subject invention, certain data may be stored in a generally accessible area and certain other data may be stored in a limited access area, where some or all of the data stored in either or both of the generally accessible areas and/or limited access areas may be raw and/or processed and/or encrypted and/or decrypted.
Devices configured to collect, receive and store at least one of static and dynamic data, where such data can be easily and securely communicated, i.e., transferred to or received from, at least one external or remote apparatus or site such as a fluid handling apparatus, a device detector, a personal computer (“PC”), and the like. The data storage element may be positioned in or on any or all of the layers of the device or associated with the device generally. See, for example, U.S. Pat. Nos. 6,238,910; 5,958,760; and 6,114,122 and U.S. Patent Publication No. US 2005/0063227 A1.
During operation, tear off frangible seals 406A, 406B, 406C, 406D are selectively openable by, for example tearing, twisting or cutting of seals 406A, 406B, 406C, 406D. This operation may be carried out by the fluid processing apparatus, for example as shown in
Reaction area compartment 444 and reaction vessel 452 may interface with fluid handling apparatus components designed to regulate the temperature of vessel contents. As an example, a reaction that could be carried out in these vessels include, but are not limited to, enzymatic treatments of a biological sample to lyse hardy microorganisms to allow access to their content. As another example, a reaction that could be carried out in these vessels include enzymatic or chemical treatments of a nucleic acid sample to shear, digest, extend, ligate, or convert the nucleic acid sample.
Air tight seals may or may not need to be created between reagent tip 442 and reaction area compartment 444. If an air tight seal is formed between reagent tip 442 and reaction area compartment 444, compression of the attached reagent compartment 404 will generate pressure which may force the fluids out through reagent tip 442, into reaction area compartment 444, through the reaction area 414 and out through reaction layer outlet 416. Passage of fluids through the reaction area 414 may result in retention of analytes in the fluid onto the reaction layer 420. Alternatively, the flow of fluids through reaction area 414 may result in the removal of bound contaminants or analytes from reaction area 414. Alternatively, reagent layer 410 in this embodiment may contain a fitting 44,8 arranged on a second surface 413, which is in fluid communication with a gaseous or reagent source 446 external to the fluid handling device 400. Air tight sealing between fitting 448 and the reaction area compartment 444 allows delivery of gases or reagents to the reaction area 414. Delivery of gases to compartment 444 may serve to drive any residual fluids in compartment 444, through reaction area 414 and reaction layer outlet 416. Fluid flow emanating from reaction layer outlet 416 can be collected into one or more fluid collection compartments (424 or 426) arranged in fluid collection layer 430. Alternatively, fittings to supply gases or reagents may be part of the operating apparatus and not part of reagent layer 410. Independent motion of the reagent layer 410, reaction layer 420 and fluid collection layer 430 allows alignment of the reaction area column to any tip in reagent layer 410 and allows alignment of the reaction area output 416 to any fluid collection compartments 424 or 426 in the fluid collection layer 430. In this exemplary embodiment, alignment of the various compartments, inlets, outlets, tip, etc. is accomplished by linear motion 428 along an axis P2 and vertical motion 429 along an axis P1 rather than rotary motion about an axis as provided for in the embodiment shown in
Reaction area compartment 544 has an inlet 515 which faces upward in communication with the bottom of reagent layer 510. Reaction area outlet 516 opens (as shown, downward) in communication with the first side of fluid collection layer 530. Reaction area compartment 544, reaction area filter 514 and reaction area outlet 516 are in fluid communication and together comprise a reaction area column. The reaction area column can be formed such that it is integral to reaction layer 520 or form a separate subcomponent which is attachable to reagent layer 520. Integrally formed components can be configured such that each component or feature that is essential or necessary for completeness is provided. Moreover, the components can be constructed such that they have a unitary construction or such that they act in a unified manner once formed. As will be appreciated by those skilled in the art, the form factor of the reaction area column depicted here is similar to commonly used spin filters for protein or nucleic acids purification but can be of other form factors.
In
If an incubation step is required, the fluid processing apparatus can provide thermal control to compartment 552 (not shown). In a similar manner, a binding buffer storable in reagent compartment 504B can be delivered and mixed with the lysate as shown in
In
The exploded view of fluid handling device 600 in
The reaction layer 620 has a reaction layer body 673 which holds reaction vessel 652, and reaction area column 644. The form factor of the reaction area column depicted here is similar to commonly used spin filters for protein or nucleic acids purification but can be of other form factors. Reaction vessel 652 and reaction area column 644 are open at the top and are in communication with the reagent compartment tips 642A, 642B, 642C, 642D, 642E. Reaction vessel 652 and reaction area column 644 are shown as detachable from reaction layer body 673.
Fluid collection layer 630 contains the waste collection compartment 626 (better shown in
The cross-sectional view of device 600 in
Reagent compartment 704A is in fluid communication with an extra long tip, 784. All of the tips, 742B, 742C, 742D, 784 may have frangible seals at their tips (not shown). Reaction layer 720 has a first surface 721 and an opposing second surface 723 arranged below reagent layer 710 in communication with an opposing second surface 713 of reagent layer 710. Reaction layer 720 contains reaction area compartment 744, reaction area 714, and reaction area output 716. Reaction area compartment 744, arranged on first surface 721, is open at the top and is in communication with an opposing second surface 713 of reagent layer 710. Reaction area output 716, arranged on second surface 723 of the reaction area 720, is open at the bottom and is in communication with fluid collection layer 730. Together, reaction area compartment 744, reaction area 714 and reaction area output 716 are in fluid communication and comprise a reaction area column. Reaction area output 716 can be placed in fluid communication with fluid collection compartment 724 in fluid collection layer 730 allowing fluid collection compartment 724 to receive fluids from 716. In some configurations, reaction layer 720 can be configured to contain a second reaction area column comprising of reaction area compartment 744′, reaction area 714′, and reaction area output 716′. Second reaction area output 716′ can be placed in fluid communication with fluid collection compartment 724′ in fluid collection layer 730 allowing fluid collection compartment 724′ to receive fluids from 716′. Independent horizontal (728 and 728′) and vertical (729) motions of the layers along axes P1 and P2 can place tip 784 into fluid communication with compartment 724 (
Reagents stored in compartments 804″ may become randomly distributed within said compartments during transport. It is desirable that these reagents be collected toward the bottom of the compartments 804″ prior to device operation.
Reagent compartments 904 can be of different shapes, for example taller or skinnier as shown in
Exemplary reagent compartments 304, 404, 504 and 604, 704, 804 and 904 are usually integrated with the rest of the device. Depending on the application and sample type, it may be desirable to use custom reagents to help develop new fluid handling processes.
Fluid processing apparatus 1260 may manipulate multiple fluid processing devices 300 at one time.
In the exemplary embodiments shown in
To further appreciate the networked configurations of multiple fluid handling devices and diagnostic devices in a communication network,
As is well understood by those skilled in the art, the Internet is a worldwide network of computer networks. Today, the Internet is a public and self-sustaining network that is available to many millions of users. The Internet uses a set of communication protocols called TCP/IP (i.e., Transmission Control Protocol/Internet Protocol) to connect hosts. The Internet has a communications infrastructure known as the Internet backbone. Access to the Internet backbone is largely controlled by Internet Service Providers (ISPs) that resell access to corporations and individuals.
The Internet Protocol (IP) enables data to be sent from one device (e.g., a phone, a Personal Digital Assistant (PDA), a computer, etc.) to another device on a network. There are a variety of versions of IP today, including, e.g., IPv4, IPv6, etc. Other IPs are no doubt available and will continue to become available in the future, any of which can, in a communication network adapted and configured to employ or communicate with one or more fluid handling devices and/or diagnostic devices, be used without departing from the scope of the invention. Each host device on the network has at least one IP address that is its own unique identifier and acts as a connectionless protocol. The connection between end points during a communication is not continuous. When a user sends or receives data or messages, the data or messages are divided into components known as packets. Every packet is treated as an independent unit of data and routed to its final destination—but not necessarily via the same path.
The Open System Interconnection (OSI) model was established to standardize transmission between points over the Internet or other networks. The OSI model separates the communications processes between two points in a network into seven stacked layers, with each layer adding its own set of functions. Each device handles a message so that there is a downward flow through each layer at a sending end point and an upward flow through the layers at a receiving end point. The programming and/or hardware that provides the seven layers of function is typically a combination of device operating systems, application software, TCP/IP and/or other transport and network protocols, and other software and hardware.
Typically, the top four layers are used when a message passes from or to a user and the bottom three layers are used when a message passes through a device (e.g., an IP host device). An IP host is any device on the network that is capable of transmitting and receiving IP packets, such as a server, a router or a workstation. Messages destined for some other host are not passed up to the upper layers but are forwarded to the other host. The layers of the OSI model are listed below. Layer 7 (i.e., the application layer) is a layer at which, e.g., communication partners are identified, quality of service is identified, user authentication and privacy are considered, constraints on data syntax are identified, etc. Layer 6 (i.e., the presentation layer) is a layer that, e.g., converts incoming and outgoing data from one presentation format to another, etc. Layer 5 (i.e., the session layer) is a layer that, e.g., sets up, coordinates, and terminates conversations, exchanges and dialogs between the applications, etc. Layer-4 (i.e., the transport layer) is a layer that, e.g., manages end-to-end control and error-checking, etc. Layer-3 (i.e., the network layer) is a layer that, e.g., handles routing and forwarding, etc. Layer-2 (i.e., the data-link layer) is a layer that, e.g., provides synchronization for the physical level, does bit-stuffing and furnishes transmission protocol knowledge and management, etc. The Institute of Electrical and Electronics Engineers (IEEE) sub-divides the data-link layer into two further sub-layers, the MAC (Media Access Control) layer that controls the data transfer to and from the physical layer and the LLC (Logical Link Control) layer that interfaces with the network layer and interprets commands and performs error recovery. Layer 1 (i.e., the physical layer) is a layer that, e.g., conveys the bit stream through the network at the physical level. The IEEE sub-divides the physical layer into the PLCP (Physical Layer Convergence Procedure) sub-layer and the PMD (Physical Medium Dependent) sub-layer.
Wireless networks can incorporate a variety of types of mobile devices, such as, e.g., cellular and wireless telephones, PCs (personal computers), laptop computers, wearable computers, cordless phones, pagers, headsets, printers, PDAs, etc. and suitable for use in a system or communication network that includes one or more diagnostic devices and/or one or more fluid handling devices. For example, mobile devices may include digital systems to secure fast wireless transmissions of voice and/or data. Typical mobile devices include some or all of the following components: a transceiver (for example a transmitter and a receiver, including a single chip transceiver with an integrated transmitter, receiver and, if desired, other functions); an antenna; a processor; display; one or more audio transducers (for example, a speaker or a microphone as in devices for audio communications); electromagnetic data storage (such as ROM, RAM, digital data storage, etc., such as in devices where data processing is provided); memory; flash memory; and/or a full chip set or integrated circuit; interfaces (such as universal serial bus (USB), coder-decoder (CODEC), universal asynchronous receiver-transmitter (UART), phase-change memory (PCM), etc.). Other components can be provided without departing from the scope of the invention.
Wireless LANs (WLANs) in which a mobile user can connect to a local area network (LAN) through a wireless connection may be employed for wireless communications between one or more diagnostic devices and/or fluid handling devices. Wireless communications can include communications that propagate via electromagnetic waves, such as light, infrared, radio, and microwave. There are a variety of WLAN standards that currently exist, such as Bluetooth®, IEEE 802.11, and the obsolete HomeRF.
By way of example, Bluetooth products may be used to provide links between mobile computers, mobile phones, portable handheld devices, personal digital assistants (PDAs), and other mobile devices and connectivity to the Internet. Bluetooth is a computing and telecommunications industry specification that details how mobile devices can easily interconnect with each other and with non-mobile devices using a short-range wireless connection. Bluetooth creates a digital wireless protocol to address end-user problems arising from the proliferation of various mobile devices that need to keep data synchronized and consistent from one device to another, thereby allowing equipment from different vendors to work seamlessly together.
An IEEE standard, IEEE 802.11, specifies technologies for wireless LANs and devices. Using 802.11, wireless networking may be accomplished with each single base station supporting several devices. In some examples, devices may come pre-equipped with wireless hardware or a user may install a separate piece of hardware, such as a card, that may include an antenna. By way of example, devices used in 802.11 typically include three notable elements, whether or not the device is an access point (AP), a mobile station (STA), a bridge, a personal computing memory card International Association (PCMCIA) card (or PC card) or another device: a radio transceiver; an antenna; and a MAC (Media Access Control) layer that controls packet flow between points in a network.
In addition, Multiple Interface Devices (MIDs) may be utilized in some wireless networks. MIDs may contain two independent network interfaces, such as a Bluetooth interface and an 802.11 interface, thus allowing the MID to participate on two separate networks as well as to interface with Bluetooth devices. The MID may have an IP address and a common IP (network) name associated with the IP address.
Wireless network devices may include, but are not limited to Bluetooth devices, WiMAX (Worldwide Interoperability for Microwave Access), Multiple Interface Devices (MIDs), 802.11x devices (IEEE 802.11 devices including, 802.11a, 802.11b and 802.11g devices), HomeRF (Home Radio Frequency) devices, Wi-Fi (Wireless Fidelity) devices, GPRS (General Packet Radio Service) devices, 3 G cellular devices, 2.5 G cellular devices, GSM (Global System for Mobile Communications) devices, EDGE (Enhanced Data for GSM Evolution) devices, TDMA type (Time Division Multiple Access) devices, or CDMA type (Code Division Multiple Access) devices, including CDMA2000. Each network device may contain addresses of varying types including but not limited to an IP address, a Bluetooth Device Address, a Bluetooth Common Name, a Bluetooth IP address, a Bluetooth IP Common Name, an 802.11 IP Address, an 802.11 IP common Name, or an IEEE MAC address.
Wireless networks can also involve methods and protocols found in, Mobile IP (Internet Protocol) systems, in PCS systems, and in other mobile network systems. With respect to Mobile IP, this involves a standard communications protocol created by the Internet Engineering Task Force (IETF). With Mobile IP, mobile device users can move across networks while maintaining their IP Address assigned once. See Request for Comments (RFC) 3344. NB: RFCs are formal documents of the Internet Engineering Task Force (IETF). Mobile IP enhances Internet Protocol (IP) and adds a mechanism to forward Internet traffic to mobile devices when connecting outside their home network. Mobile IP assigns each mobile node a home address on its home network and a care-of-address (CoA) that identifies the current location of the device within a network and its subnets. When a device is moved to a different network, it receives a new care-of address. A mobility agent on the home network can associate each home address with its care-of address. The mobile node can send the home agent a binding update each time it changes its care-of address using Internet Control Message Protocol (ICMP).
In basic IP routing (e.g., outside mobile IP), routing mechanisms rely on the assumptions that each network node always has a constant attachment point to the Internet and that each node's IP address identifies the network link it is attached to. Nodes include a connection point, which can include a redistribution point or an end point for data transmissions, and which can recognize, process and/or forward communications to other nodes. For example, Internet routers can look at an IP address prefix or the like identifying a device's network. Then, at a network level, routers can look at a set of bits identifying a particular subnet. Then, at a subnet level, routers can look at a set of bits identifying a particular device. With typical mobile IP communications, if a user disconnects a mobile device from the Internet and tries to reconnect it at a new subnet, then the device has to be reconfigured with a new IP address, a proper netmask and a default router. Otherwise, routing protocols would not be able to deliver the packets properly.
Computing system 1500, described above, can be deployed as part of a computer network that includes one or more diagnostic devices and/or fluid handling devices. In general, the above description for computing environments applies to both server computers and client computers deployed in a network environment.
In operation, a user (not shown) may interact with a computing application running on a client computing environment to obtain desired data and/or computing applications. The data and/or computing applications may be stored on server computing environment 1500 and communicated to cooperating users through client computing environments over exemplary communications network 1550. A participating user may request access to specific data and applications housed in whole or in part on server computing environment 1500. These data may be communicated between client computing environments and server computing environments for processing and storage. Server computing environment 1500 may host computing applications, processes and applets for the generation, authentication, encryption, and communication data and applications and may cooperate with other server computing environments (not shown), third party service providers (not shown), network attached storage (NAS) and storage area networks (SAN) to realize application/data transactions.
Methods according to this disclosure include purification and concentration of DNA from a sample through a silica filter. Commercial kits, such as the Qiagen DNeasy®Blood and Tissue Kits (Qiagen 69504) are commonly used for this purpose. A pilot experiment was performed in order to test whether compression of a collapsible reagent compartment and application of an external pressure source is sufficient to perform such a purification.
For a control Qiagen purification, 200 uL of ˜2.5 ng/uL human genomic DNA sample was mixed with 200 uL of Buffer AL and 200 uL EtOH. This mixture was applied to the silica column and processed by centrifugation per manufacturer's instruction. Each sample was washed with 500 uL each of buffer AW1 and AW2 followed by elution with 100 uL Buffer AE.
The same reagents were used to test the invention, except that the process was modified in the following manner. The sample mixtures, and the washes were passed through the column, not by centrifugation, but by pressure fit sealing of a standard laboratory transfer pipette to the silica column and compressing the pipette bulb. The last wash was dried by application of ˜15 psi air through the column for 3 minutes. The elution buffer was also passed through the column with 15 psi air for approximately 15 seconds. Four control and prototype purifications were performed each. The amount of DNA in each eluate was quantitated by quantitative polymerase chain reaction (PCR) using primers designed for the rnaseP gene. The data is shown in
A ZR Genomic DNA II Kit™ from Zymo Research (Orange, Calif.) can be used to purify DNA from human blood. Purified DNA from blood can be used in downstream analytical process to provide valuable information such as determination of the genetic relationships between individuals, assessment of a patient's likely response to a therapeutic, and the identification of possible infectious agents.
To test the invention, a device similar to device 500 shown in
Evaluation of commercially available rapid influenza diagnostic tests (RIDTs) by the CDC (Evaluation of Rapid Influenza Diagnostic Testes for Detection of Novel Influenza A (H1N1) Virus. Morbidity and Mortality Weekly Report. 58(30): 826-829. Aug. 7, 2009) revealed shortcomings with the ability of these tests to detect the H1N1 strain of influenza. These tests miss many cases of the H1N1 virus, with a detection rate of only 40-69% and none of these tests can distinguish between the different strains. In this study, the CDC used purification of nucleic acids from the samples followed by rRT-PCR assays (5′ fluorogenic exonuclease assay) as the “gold standard” to detect the presence of influenza. The rRT-PCR tests used by the CDC are well over 99% sensitive and can distinguish between the various influenza strains. While these types of rRT-PCR tests are available to the general public, they typically require that the sample be sent to a central laboratory for testing. The requirement for well trained personnel, at well equipped laboratories, at locations remote from the patient to perform these tests increases the cost of these tests. Transport of the sample to these laboratories can lead to sample degradation and places a requirement for sample stabilization. In addition, the delays (days or weeks) in reporting the results back to the doctor and patient reduces the utility of the information. Using the inventions described above, these highly sensitive tests can be performed at the doctor's office safely, and in a time and cost effective manner. By way of example, a sample from the patient (for example nasal swabs) is collected and placeable into a reaction compartment 552 of device 500 as described in
Rapid identification of potential bacterial infections is a growing need. For example, Clostridium difficile infection (CDI) has been cited by the CDC as an emerging threat. C. difficile is a spore forming bacillus bacteria that can infect the elderly and patients with weakened immune systems. Patients treated with antibiotics for other infections are also at elevated risk for CDI. Infection with C. difficile can result in Colitis, other intestinal conditions, sepsis and death. Both the rates of CDI and the severity of symptoms from these infections have been rising in recent years and it is believed that the emergence of more virulent strains is, in part, to blame. Since the majority of human cases of CDI occur in association with inpatient stays in hospitals or long-term care facilities, technologies to rapidly detect CDI and to identify the underlying strains is of great interest. MDx is ideally suited for this role.
C. difficile is an example of a target with hardy cell walls that must be breached to release the nucleic acids for purification and detection. By way of example, different configuration of the invention can be utilized in the analysis of C. difficile containing samples. As a first example, samples suspected of containing C. difficile can be added to a septum containing reagent compartment with lysis buffer and glass beads similar to that described in
As a second example, a fluid handling device can be used that has an integrated reaction vessel in the reaction layer, a second detachable reaction vessel in the reaction layer and a detachable elution vessel in the fluid collection layer. The sample can be added to the integrated reaction vessel. The user selects a detachable reaction vessel that contains reagents (for example lyophilized enzymes) optimized for degradation of the C. difficile cell wall and attaches it to the reaction layer. The user also selects a detachable elution vessel that contains reagents (for example buffers, enzymes, primers and probes) optimized for detection of C. difficile and attaches it to the fluid collection layer. Both the detachable reaction and elution vessel may be sealed with a temporary Aluminum lid seal for storage and transport. The option of selecting and attaching detachable reaction and elution vessels allows the user to select reagents optimized for their sample target. These detachable reaction and elution vessels may be supplied as part of a kit with all necessary components or may be ordered separately as needed. Once the user had attached the required reaction and and elution vessel to the device and added the sample to the integrated reaction vessel on the device, the user places the device into the fluid processing apparatus and processes the sample as described previously.
Kits are also contemplated as an aspect of the invention. Suitable kits for extracting nucleic acid from a sample, include, for example, a device with prefilled reagent compartments packaged in an hermitically sealed pouch. The user opens the pouch and inserts the device into an fluid processing apparatus capable of running the fluid handling protocol. Kits may be differentiated by one or more of the following:
Devices are manufacturable using one or more of vacuum, pressure, thermal forming, blow molding, and injection molding processes. Resins used in these processes will depend for example, on the reagents being packaged and on the size of the manufactured device. Reagent compartments that contain solid or liquid reagents are manufacturable with blow, fill, seal and modified blow, fill, seal manufacturing techniques. Reagent compartments may be manufactured individually or in ganged configurations. Existing off-the-shelf components for reagent compartments, reaction vessels, eluate collection vessels may be integrated into the final device using custom holders for the parts. Alternatively the device design may be entirely custom with no off-the-shelf parts. The device may consist of three layers with independent motion or one or more layers may be integrated together and may be desirable to produce lower cost devices. Devices may be manufactured that combine a fluid handling device with detection device. Other methods of manufacturing can be used without departing from the scope of the invention.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Application No. 61/141,645, filed Dec. 30, 2008, and Application No. 61/166,760, filed Apr. 5, 2009, which applications are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/69812 | 12/30/2009 | WO | 00 | 7/26/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61166760 | Apr 2009 | US | |
| 61141645 | Dec 2008 | US |
