Patent Publication Number: US-2010129827-A1

Title: Method and device for sample preparation control

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates generally to nucleic acid assays and, more particularly, to a device and method for preparing a sample for nucleic acid amplification and for verifying the integrity of the sample preparation process. 
     Methods for amplifying nucleic acids provide useful tools for the detection of human pathogens, detection of human genetic polymorphisms, detection of RNA and DNA sequences, for molecular cloning, sequencing of nucleic acids, and the like. In particular, the polymerase chain reaction (PCR) has become an important tool in the cloning of DNA sequences, forensics, paternity testing, pathogen identification, disease diagnosis, and other useful methods where the amplification of a nucleic acid sequence is desired. See e.g.,  PCR Technology: Principles and Applications for DNA Amplification  (Erlich, ed., 1992);  PCR Protocols: A Guide to Methods and Applications  (Innis et al., eds, 1990). 
     The analysis of samples suspected of containing a nucleic acid sequence of interest generally involves a series of sample preparation steps, which may include filtration, cell lysis, nucleic acid purification, and mixing with reagents. To be confident about the results of a nucleic acid assay, it would be useful to control for the integrity of the sample preparation process. The present invention addresses this and other problems. 
     SUMMARY 
     According to one aspect, the invention provides a method for preparing a sample for a nucleic acid amplification reaction and for verifying the effectiveness of the sample preparation. The sample is suspected of containing target entities selected from the group consisting of cells, spores, microorganisms, and viruses, and the target entities comprise at least one target nucleic acid sequence. The method comprises the step of introducing the sample into a device having a mixing chamber for mixing the sample with sample preparation controls. The sample preparation controls are selected from the group consisting of cells, spores, microorganisms, and viruses, and the sample preparation controls comprise a marker nucleic acid sequence. The device further has a lysing chamber and a reaction chamber. The sample is mixed with the sample preparation controls in the mixing chamber. The method further comprises the steps of subjecting the sample preparation controls and the target entities, if present in the sample, to a lysis treatment in the lysing chamber, subjecting nucleic acid released in the lysing chamber to nucleic acid amplification conditions in the reaction chamber, and detecting the presence or absence of the at least one target nucleic acid sequence and of the marker nucleic acid sequence. Positive detection of the marker nucleic acid sequence indicates that the sample preparation process was satisfactory, while the inability to detect the marker nucleic acid sequence indicates inadequate sample preparation. 
     In some embodiments, the lysing chamber contains solid phase material, and the method further comprises the step of forcing the sample mixed with the sample preparation controls to flow through the lysing chamber to capture the sample preparation controls and the target entities, if present in the sample, with the solid phase material prior to the lysis treatment. In some embodiments, the solid phase material comprises at least one filter having a pore size sufficient to capture the sample preparation controls and the target entities. The sample may be pre-filtered (e.g., to remove coarse material) prior to mixing the sample with the sample preparation controls. In some embodiments, the lysis treatment comprises subjecting the sample preparation controls and the target entities to ultrasonic energy using an ultrasonic transducer coupled to a wall of the lysing chamber. The lysis treatment may optionally comprise agitating beads in the lysing chamber. In some embodiments, the sample preparation controls are spores. In some embodiments, the mixing step comprises dissolving a dried bead containing the sample preparation controls. In some embodiments, the lysis treatment comprises contact with a chemical lysis agent. In some embodiments, the nucleic acid amplification conditions comprise polymerase chain reaction (PCR) conditions. In some embodiments, the presence or absence of the marker nucleic acid sequence is detected by determining if a signal from a probe capable of binding to the marker nucleic acid sequence exceeds a threshold level. 
     According to another aspect, the invention provides a device for preparing a sample for a nucleic acid amplification reaction and for verifying the effectiveness of the sample preparation. The sample is suspected of containing target entities selected from the group consisting of cells, spores, microorganisms, and viruses, and the target entities comprise at least one target nucleic acid sequence. The device comprises a body having a first chamber containing sample preparation controls to be mixed with the sample. The sample preparation controls are selected from the group consisting of cells, spores, microorganisms, and viruses, and the sample preparation controls comprise a marker nucleic acid sequence. The body also has a lysing chamber for subjecting the sample preparation controls and the target entities, if present in the sample, to a lysis treatment to release the nucleic acid therefrom. The body further has a reaction chamber for holding the nucleic acid for amplification and detection. The device further comprises at least one flow controller for directing the sample mixed with the sample preparation controls to flow from the first chamber into the lysing chamber and for directing the nucleic acid released in the lysing chamber to flow into the reaction chamber. The device further contains primers and probes for amplifying and detecting the marker nucleic acid sequence and the at least one target nucleic acid sequence. 
     In some embodiments, the lysing chamber contains solid phase material for capturing the sample preparation controls and the target entities, if present in the sample, as the sample flows through the lysing chamber, the device further includes at least one waste chamber for receiving used sample fluid that has flowed through the lysing chamber, and the at least one flow controller is further capable of directing used sample fluid that has flowed through the lysing chamber to flow into the waste chamber. In some embodiments, the solid phase material comprises at least one filter having a pore size sufficient to capture the sample preparation controls and the target entities. In some embodiments, the device further comprises an ultrasonic transducer coupled to a wall of the lysing chamber to sonicate the lysing chamber. In some embodiments, the device further comprises beads in the lysing chamber for rupturing the sample preparation controls and the target entities. In some embodiments, the sample preparation controls are spores. In some embodiments, the sample preparation controls are in a dried bead that is dissolvable in liquid. In some embodiments, the primers and probes are in a dried bead in the reaction chamber, the bead being dissolvable in liquid. In some embodiments, the body includes a mixing chamber connected to the reaction chamber, and the primers and probes are in a dried bead in the mixing chamber, the bead being dissolvable in liquid. 
     According to another aspect, the present invention provides a method for determining the effectiveness of a lysis procedure. The method comprises the steps of mixing sample preparation controls with a sample suspected of containing target entities selected from the group consisting of cells, spores, microorganisms, and viruses. The target entities comprise at least one target nucleic acid sequence. The sample preparation controls are selected from the group consisting of cells, spores, microorganisms, and viruses, and the sample preparation controls comprise a marker nucleic acid sequence. The mixture of the sample preparation controls and the target entities, if present in the sample, are subjected to a lysis treatment. The method further comprises the steps of detecting the presence or absence of the marker nucleic acid sequence to determine if nucleic acid was released from the sample preparation controls during the lysis treatment. Positive detection of the marker nucleic acid sequence indicates satisfactory lysis, while the inability to detect the marker nucleic acid sequence indicates inadequate lysis. 
     In some embodiments, the method further comprises the step of forcing the sample mixed with the sample preparation controls to flow through a chamber containing solid phase material to capture the sample preparation controls and the target entities, if present in the sample, with the solid phase material prior to the lysis treatment. In some embodiments, the solid phase material comprises at least one filter having a pore size sufficient to capture the sample preparation controls and the target entities. In some embodiments, the sample is pre-filtered prior to mixing the sample with the sample preparation controls. In some embodiments, the lysis treatment comprises subjecting the sample preparation controls and the target entities to ultrasonic energy. The lysis treatment may also comprise agitating beads to rupture the sample preparation controls and the target entities. In some embodiments, the sample preparation controls are spores. In some embodiments, the mixing step comprises dissolving a dried bead containing the sample preparation controls. In some embodiments, the lysis treatment comprises contact with a chemical lysis agent. In some embodiments, the marker nucleic acid sequence is detected by amplifying the marker nucleic acid sequence (e.g., by PCR) and detecting the amplified marker nucleic acid sequence. In some embodiments, the amplified marker nucleic acid sequence is detected by determining if a signal from a probe capable of binding to the marker nucleic acid sequence exceeds a threshold level. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of the fluid control and processing device according to an embodiment of the present invention; 
         FIG. 2  is another perspective view of the device of  FIG. 1 ; 
         FIG. 3  is an exploded view of the device of  FIG. 1 ; 
         FIG. 4  is an exploded view of the device of  FIG. 2 ; 
         FIG. 5  is an elevational view of a fluid control apparatus and gasket in the device of  FIG. 1 ; 
         FIG. 6  is a bottom plan view of the fluid control apparatus and gasket of  FIG. 5 ; 
         FIG. 7  is a top plan view of the fluid control apparatus and gasket of  FIG. 5 ; 
         FIG. 8  is a cross-sectional view of the rotary fluid control apparatus of  FIG. 7  along  8 - 8 ; 
       FIGS.  9 A- 9 LL are top plan views and cross-sectional views illustrating a specific protocol for controlling and processing fluid using the fluid control and processing device of  FIG. 1 ; 
         FIG. 10  is a cross-sectional view of a piston assembly; and 
         FIG. 11  is a cross-sectional view of a side-filtering chamber. 
     
    
    
     DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
       FIGS. 1-4  show a fluid control and processing system  10  including a housing  12  having a plurality of chambers  13 .  FIG. 1  shows the chambers  13  exposed for illustrative purposes. A top cover will typically be provided to enclose the chambers  13 . As best seen in  FIGS. 3 and 4 , a fluid control device  16  and a reaction vessel  18  are connected to different portions of the housing  12 . The fluid control device in the embodiment shown is a rotary fluid control valve  16 . The valve  16  includes a valve body  20  having a disk portion  22  and a tubular portion  24 . The disk portion  22  has a generally planar external port surface  23 , as best seen in  FIG. 3 . The valve  16  is rotatable relative to the housing  12 . The housing  12  includes a plurality of chamber ports  25  facing the external port surface  23  of the disk portion  22  of the valve  16  ( FIG. 4 ) to permit fluidic communication between the chambers  13  and the valve  16 . An optional seal or gasket  26  is disposed between the disk portion  22  and the housing  12 . The disk portion  22  further includes a filter  27  and an outer wall  28 , and a toothed periphery  29 . 
     As seen in  FIG. 4 , the disk portion  22  includes a lysing chamber  30 . The lysing chamber  30  may contain solid phase material for capturing cells, spores, viruses, or microorganisms to be lysed. Suitable solid phase materials include, without limitation, filters, beads, fibers, membranes, filter paper, glass wool, polymers, or gels. In a specific embodiment, the solid phase material is a filter having a pore size sufficient to capture target cells, spores, viruses, or microorganisms to be lysed. 
     As shown in  FIGS. 5-8 , the outer wall  28  encloses the lysing chamber  30  and the bottom end of the disk portion  22  of the valve  16 . In  FIG. 8 , the lysing chamber  30  includes a first fluid processing port  32  coupled to a first fluid processing channel  34 , and a second fluid processing port  36  coupled to a second fluid processing channel  38 . The first fluid processing channel  34  is coupled to a first outer conduit  40  ending at a first external port  42  at the external port surface  23 , while the second fluid processing channel  38  is coupled to a second outer conduit  44  ending at a second external port  46  at the external port surface  23 . A fluid displacement channel  48  is coupled to the first fluid processing channel  34  and first conduit  40  near one end, and to a fluid displacement chamber  50  at the other end. The first outer conduit  40  serves as a common conduit for allowing fluidic communication between the first external port  42  and either or both of the first fluid processing channel  34  and the fluid displacement channel  48 . The lysing chamber  30  is in continuous fluidic communication with the fluid displacement chamber  50 . 
     As shown in  FIGS. 6-8 , the external ports  42 ,  46  are angularly spaced from one another relative to the axis  52  of the valve  16  by about 180°. The external ports  42 ,  46  are spaced radially by the same distance from the axis  52 . The axis  52  is perpendicular to the external port surface  23 . In another embodiment, the angular spacing between the external ports  42 ,  46  may be different. The configuration of the channels in the disk portion  22  may also be different in another embodiment. For example, the first fluid processing channel  34  and the first outer conduit  40  may be slanted and coupled directly with the fluid displacement chamber  50 , thereby eliminating the fluid displacement channel  48 . The second fluid displacement channel  38  may also be slanted and extend between the second fluid processing port  36  and the second external port  46  via a straight line, thereby eliminating the second outer conduit  44 . In addition, more channels and external ports may be provided in the valve  16 . As best seen in  FIG. 3 , a crossover channel or groove  56  is desirably provided on the external port surface  23 . The groove  56  is curved and desirably is spaced from the axis  52  by a constant radius. In one embodiment, the groove  56  is a circular arc lying on a common radius from the axis  52 . As discussed in more detail below, the groove  56  is used for filling the vessel. 
     As shown in  FIG. 8 , the fluid displacement chamber  50  is disposed substantially within the tubular portion  24  of the valve  16  and extends partially into the disk portion  22 . A fluid displacement member in the form of a plunger or piston  54  is movably disposed in the chamber  50 . When the piston  54  moves upward, it expands the volume of the chamber  50  to produce suction for drawing fluid into the chamber  50 . When the piston  54  moves downward, it decreases the volume of the chamber  50  to drive fluid out of the chamber  50 . 
     As the rotary valve  16  is rotated around its axis  52  relative to the housing  12  of  FIGS. 1-4 , one of the external ports  42 ,  46  may be open and fluidicly coupled with one of the chambers  13  or reaction vessel  18 , or both external ports  42 ,  46  may be blocked or closed. In this embodiment, at most only one of the external ports  42 ,  46  is fluidicly coupled with one of the chambers or reaction vessel  18 . Other embodiments may be configured to permit both external ports  42 ,  46  to be fluidicly coupled with separate chambers or the reaction vessel  18 . Thus, the valve  16  is rotatable with respect to the housing  12  to allow the external ports  42 ,  46  to be placed selectively in fluidic communication with a plurality of chambers which include the chambers  13  and the reaction vessel  18 . Depending on which external port  42 ,  46  is opened or closed and whether the piston  54  is moved upward or downward, the fluid flow in the valve  16  can change directions, the external ports  42 ,  46  can each switch from being an inlet port to an outlet port, and the fluid flow may pass through the processing region  30  or bypass the lysing chamber  30 . In a specific embodiment, the first external port  42  is the inlet port so that the inlet side of the lysing chamber  30  is closer to the fluid displacement chamber  54  than the outlet side of the lysing chamber  30 . 
     FIGS.  9 A- 9 LL illustrate the operation of the valve  16  for conducting a nucleic acid assay of a sample suspected of containing one or more target entities (e.g., cells, spores, viruses, or microorganisms). The target entities comprise at least one target nucleic acid sequence for which the sample is being tested. A sample may be introduced into the housing  12  of the fluid control and processing device  10 , which may be configured as a cartridge, by a variety of mechanisms, manual or automated. For manual addition, a measured volume of material may be placed into a receiving area of the housing  12  (e.g., one of the plurality of chambers) through an input port and a cap is then placed over the port. Alternatively, the receiving area may be covered by a rubber or similar barrier and the sample is injected into the receiving area by puncturing the barrier with a needle and injecting the sample through the needle. Alternatively, a greater amount of sample material than required for the analysis can be added to the housing  12  and mechanisms within the housing  12  can effect the precise measuring and aliquoting of the sample needed for the specified protocol. 
     It may be desirable to place certain samples, such as tissue biopsy material, soil, feces, exudates, and other complex material into another device or accessory and then place the secondary device or accessory into the housing causing a mechanical action which effects a function such as mixing, dividing, or extraction. For example, a piece of tissue may be placed into the lumen of a secondary device that serves as the input port cap. When the cap is pressed into the port, the tissue is forced through a mesh that slices or otherwise divides the tissue. 
     For automated sample introduction, additional housing or cartridge design features are employed and, in many cases, impart sample collection functionality directly into the housing. With certain samples, such as those presenting a risk of hazard to the operator or the environment, such as human retrovirus pathogens, the transfer of the sample to the housing may pose a risk. Thus, in one embodiment, a syringe or sipper may be integrated into the device to provide a means for moving a sample directly into the housing. Alternatively, the device may include a venous puncture needle and a tube forming an assembly that can be used to acquire a sample. After collection, the tube and needle are removed and discarded, and the housing  12  is then placed in an instrument to effect processing. The advantage of such an approach is that the operator or the environment is not exposed to pathogens. 
     The input port can be designed with a consideration of appropriate human factors as a function of the nature of the intended specimen. For example, respiratory specimens may be acquired from the lower respiratory tract as expectorants from coughing. Swab or brush samples may also be placed into the device. In the former case, the input port can be designed to allow the patient to cough directly into the housing  12  or to otherwise facilitate spitting of the expectorated sample into the housing. For brush or swab samples, the brush or swab is preferably placed in one of the chambers of the device  10  and the sample is eluted off the brush or swab using, e.g., water or other suitable elution fluid. In addition, the housing  12  may include features that facilitate the breaking off and retaining of the end of the swab or brush in the sample-receiving chamber. 
     In another embodiment, the housing  12  includes one or more input tubes or sippers that may be positioned in a sample pool so that the sample material flows into the housing  12 . Alternatively, a hydrophilic wicking material can function to draw a sample into the device. For example, the entire cartridge can be immersed directly into the sample, and a sufficient amount of sample is absorbed into the wicking material and wicks into the housing  12 . The housing is then removed, and can be transported to the laboratory or analyzed directly using a portable instrument. In another embodiment, tubing can be utilized so that one end of the tube is in direct communication with the housing to provide a fluidic interface with at least one chamber and the other end is accessible to the external environment to serve as a receiver for sample. The tube can then be placed into a sample and serve as a sipper. Thus, the device may include a variety of features for collecting a sample from various different sources and for moving the sample into the housing  12 , thereby reducing handling and inconvenience. 
     In FIGS.  9 A and  9 AA, a sample is placed in a mixing chamber  60 , e.g., by pipetting, and then a lid is placed over the chamber  60 . The sample will be tested to determine if it contains one or more target nucleic acid sequences. This requires sample preparation steps, e.g., lysing the target cells, spores, viruses, or microorganisms containing the target nucleic acid sequence. The chamber  60  contains sample preparation controls to be mixed with the sample. The sample preparation controls are also cells, spores, viruses, or microorganisms. The sample preparation controls contain a marker nucleic acid sequence different than the target nucleic acid sequence for which the sample is being assayed. The marker nucleic acid sequence will be detected in the reaction chamber  18  later in the assay, along with the target nucleic acid sequence if the target nucleic acid sequence is present in the sample. In order for the marker nucleic acid sequence to be detected, the sample preparation controls must be successfully lysed to release their nucleic acid and the nucleic acid must be successfully mixed with amplification reagents and amplified. The sample preparation controls thus indicate that sample preparation was adequate for the nucleic acid assay if they can be detected and inadequate if they cannot be detected. The sample preparation controls thus verify that the sample preparation was effective if they can be positively detected, so that one can feel confident in the assay results. 
     In one preferred embodiment, the sample preparation controls are spores containing a specific marker nucleic acid sequence to be amplified and detected. For example, 2,000 to 10,000 spores containing a specific marker nucleic acid sequence are generally preferred, and more preferably about 6,000 spores are used as the sample preparation controls. The spores should be cleaned so that there is no external nucleic acid in order to prove that lysis step of the sample preparation is effective, and not just loosening external nucleic acid. In addition, the sample preparation controls are preferably stored in one of the chambers of the housing  12  in a lyophilized or dried-down bead that is quickly dissolvable in liquid. Methods for making such beads are well known in the art and are described in U.S. Pat. No. 5,593,824 and in co-pending U.S. patent application Ser. No. 10/672,266 filed Sep. 25, 2003, the disclosures of which are incorporated by reference herein. 
     The sample suspected of containing target cells, spores, viruses, or microorganisms is mixed with the sample preparation controls in the chamber  60 . The mixing is preferably accomplished by dissolving a dried bead containing the sample preparation controls in the sample fluid. The first external port  42  is placed in fluidic communication with the chamber  60  by rotating the valve  16 , and the piston  54  is pulled upward to draw a fluid sample from the chamber  60  through the first outer conduit  40  and fluid displacement channel  48  to the fluid displacement chamber  50 , bypassing the lysing chamber  30 . For simplicity, the piston  54  is not shown in FIGS.  9 A- 9 LL. The valve  16  is then rotated to place the second external port  46  in fluidic communication with a waste chamber  64  as shown in FIGS.  9 B and  9 BB. The piston  54  is pushed downward to drive the fluid sample mixed with the sample preparation controls through the lysing chamber  30  to the waste chamber  64 . In a specific embodiment, the lysing chamber  30  includes at least one filter  27  having a pore size sufficient for capturing the target cells, spores, viruses, or microorganisms, if present in the sample, as well as capturing the sample preparation controls, as the sample fluid passes through the lysing chamber  30 . For this reason, it is desirable that the sample preparation controls have the same approximate size or be slightly smaller than the target cells, spores, viruses, or microorganisms in the sample to prove that the filtration of the target entities, if they were present in the sample, was successful. In alternative embodiments, other solid phase materials may be provided in the lysing chamber  30 . 
     In FIGS.  9 C and  9 CC, the valve  16  is rotated to place the first external port  42  in fluidic communication with a wash chamber  66 , and the piston  54  is pulled upward to draw a wash fluid from the wash chamber  66  into the fluid displacement chamber  50 , bypassing the lysing chamber  30 . The valve  16  is then rotated to place the second external port  46  in fluidic communication with the waste chamber  64  as shown in FIGS.  9 D and  9 DD. The piston  54  is pushed downward to drive the wash fluid through the lysing chamber  30  to the waste chamber  64 . The above washing steps may be repeated as desired. The intermediate washing is used to remove unwanted residue within the valve  16 . 
     In FIGS.  9 E and  9 EE, the valve  16  is rotated to place the first external port  42  in fluidic communication with a buffer chamber  70 , and the piston  54  is pulled upward to draw a lysis buffer (e.g., water or water mixed with lysing agents) from the buffer chamber  70  into the fluid displacement chamber  50 , bypassing the lysing chamber  30 . The valve  16  is then rotated to place the second external port  46  in fluidic communication with the waste chamber  64  as shown in FIGS.  9 F and  9 FF. The piston  54  is pushed downward to drive the buffer fluid into the lysing chamber  30 . In  FIGS. 9G , and  9 GG, the valve  16  is rotated to close the external ports  42 ,  46 . 
     The sample preparation controls and the target cells, viruses, spores, or microorganisms, if present, are subjected to a lysis treatment in the lysing chamber  30 . The purpose of the lysis treatment is to break the outer walls of the sample preparation controls and of the target cells, viruses, spores, or microorganisms, if present, to release their nucleic acid. The sample preparation controls are preferably the same level of difficulty or more difficult to lyse than the target cells, viruses, spores, or microorganisms to prove that the lysis treatment was effective. Liberation of nucleic acids from the cells, viruses, spores, or microorganisms, and denaturation of DNA binding proteins may generally be performed by chemical, physical, or electrolytic lysis methods. For example, chemical methods generally employ lysing agents to disrupt the cells and extract the nucleic acids from the cells, followed by treatment of the extract with chaotropic salts such as guanidinium isothiocyanate or urea to denature any contaminating and potentially interfering proteins. Where chemical extraction and/or denaturation methods are used, the appropriate lysing agents are preferably in the lysis buffer stored in the chamber  70  and pumped into the lysing chamber  30 . 
     Alternatively, physical methods may be used to extract the nucleic acids and denature DNA binding proteins. U.S. Pat. No. 5,304,487, incorporated herein by reference in its entirety for all purposes, discusses the use of physical protrusions within microchannels or sharp edged particles within a chamber or channel to pierce cell membranes and extract their contents. Combinations of such structures with piezoelectric elements for agitation can provide suitable shear forces for lysis. More traditional methods of cell extraction may also be used, e.g., employing a channel with restricted cross-sectional dimension which causes cell lysis when the sample is passed through the channel with sufficient flow pressure. Alternatively, cell extraction and denaturing of contaminating proteins may be carried out by applying an alternating electrical current. A variety of other methods may be utilized within the device of the present invention to effect cell lysis/extraction, including, e.g., subjecting cells to ultrasonic agitation, or forcing cells through microgeometry apertures, thereby subjecting the cells to high shear stress resulting in rupture. 
     In one preferred embodiment, the lysis treatment comprises sonicating the lysing chamber  30  using an ultrasonic transducer  76  coupled to the outer wall  28  of the lysing chamber  30 . The ultrasonic transducer  76 , preferably an ultrasonic horn, is placed in contact with the wall  28  to transmit ultrasonic energy into the lysing chamber  30  to facilitate lysing of the cells, spores, viruses, or microorganisms. Suitable ultrasonic horns are commercially available from Sonics &amp; Materials, Inc. having an office at 53 Church Hill, Newton, Conn. 06470-1614, U.S.A. Alternatively, the ultrasonic transducer may comprise a piezoelectric disk or any other type of ultrasonic transducer that may be coupled to the wall  28 . In addition, beads (e.g., glass or polystyrene beads) are preferably agitated in the lysing chamber  30  to rupture the cells, spores, viruses, or microorganisms. The pressure waves or pressure pulses created by the transducer  76  vibrating against the wall  28  causes the beads to move in ballistic motion in the lysis buffer and cause the rupturing. In these embodiments employing an ultrasonic transducer, the lysis buffer should be an ultrasonic transmission medium, e.g., deionized water. The lysis buffer may also include one or more lysing agents to aid in the lysis. In the presently preferred embodiment, the wall  28  is a deflectable plastic wall as described in co-pending U.S. patent application Ser. No. 09/972,221 filed Oct. 4, 2001 the disclosure of which is incorporated by reference herein. 
     In FIGS.  9 H and  9 HH, the valve  16  is rotated to place the second external port  46  in fluidic communication with a reagent chamber  78 , and the piston  54  is pushed downward to elute the lysate in the lysing chamber  30  to the reagent chamber  78 . The reagent chamber  78  preferably contains all of the necessary nucleic acid amplification reagents and probes (e.g., enzyme, primers, and fluorescent probes) to amplify and detect the marker nucleic acid sequence of the sample preparation controls and the one or more target nucleic acid sequences for which the sample is being tested. Any excess lysate is dispensed into the waste chamber  64  via the second external port  46  after rotating the valve  16  to place the port  46  in fluidic communication with the waste chamber  64 , as shown in FIGS.  9 I and  9 II. The lysate containing nucleic acid released in the lysing chamber  30  is then mixed in the reagent chamber  78 . This is carried out by placing the fluid displacement chamber  50  in fluidic communication with the reagent chamber  78  as shown in FIGS.  9 J and  9 JJ, and moving the piston  54  up and down. Toggling of the mixture through the filter in the processing region  30 , for instance, allows larger particles trapped in the filter to temporarily move out of the way to permit smaller particles to pass through. 
     The reagents and probes for amplifying and detecting the marker nucleic acid sequence of the sample preparation controls and the one or more target nucleic acid sequences for which the sample is being tested are preferably stored in chamber  78  in a lyophilized or dried-down bead that is quickly dissolvable in liquid. Methods for making such beads are well known in the art and are described in U.S. Pat. No. 5,593,824 and in co-pending U.S. patent application Ser. No. 10/672,266 filed Sep. 25, 2003, the disclosures of which are incorporated by reference herein. In an alternative embodiment, the reagents and probes are stored in the reaction chamber of the reaction vessel  18 . 
     In  FIGS. 9K ,  9 KK, and  9 K′K′, the valve  16  is rotated to place the first external port  42  in fluidic communication with a first branch  84  coupled to the reaction vessel  18 , while the second branch  86  which is coupled to the reaction vessel  18  is placed in fluidic communication with the crossover groove  56 . The first branch  84  and second branch  86  are disposed at different radii from the axis  52  of the valve  16 , with the first branch  84  having a common radius with the first external port  42  and the second branch  86  having a common radius with the crossover groove  56 . The crossover groove  56  is also in fluidic communication with the reagent chamber  78  ( FIG. 9K ), and serves to bridge the gap between the reagent chamber  78  and the second branch  86  to provide crossover flow therebetween. The external ports are disposed within a range of external port radii from the axis and the crossover groove is disposed within a range of crossover groove radii from the axis, where the range of external port radii and the range of crossover groove radii are non-overlapping. Placing the crossover groove  56  at a different radius from the radius of the external ports  42 ,  46  is advantageous because it avoids cross-contamination of the crossover groove  56  by contaminants that may be present in the area near the surfaces between the valve  16  and the housing  12  at the radius of the external ports  42 ,  46  as a result of rotational movement of the valve  16 . Thus, while other configurations of the crossover groove may be used including those that overlap with the radius of the external ports  42 ,  46 , the embodiment as shown is a preferred arrangement that isolates the crossover groove  56  from contamination from the area near the surfaces between the valve  16  and the housing  12  at the radius of the external ports  42 ,  46 . 
     To fill the reaction vessel  18 , the piston  54  is pulled upward to draw the reaction mixture in the reagent chamber  78  through the crossover groove  56  and the second branch  86  into the reaction vessel  18 . The valve  16  is then rotated to place the second external port  46  in fluidic communication with the first branch  84  and to close the first external port  42 , as shown in FIGS.  9 L and  9 LL. The piston  54  is pushed downward to pressurize the reaction mixture inside the reaction vessel  18 . The reaction vessel  18  has a reaction chamber for holding the reaction mixture for nucleic acid amplification and detection. The reaction chamber may be inserted into a thermal reaction sleeve for performing nucleic acid amplification and detection. The two branches  84 ,  86  allow filling and evacuation of the reaction chamber of the reaction vessel  18 . The vessel maybe connected to the housing  12  by ultrasonic welding, mechanical coupling, or the like, or be integrally formed with the housing  12  such as by molding. 
     The reaction mixture in the reaction chamber of the vessel  18  is subjected to nucleic acid amplification conditions. Amplification of an RNA or DNA template using reactions is well known (see U.S. Pat. Nos. 4,683,195 and 4,683,202 ; PCR Protocols: A Guide to Methods and Applications  (Innis et al., eds, 1990)). Methods for amplifying and detecting nucleic acids by PCR using a thermostable enzyme are disclosed in U.S. Pat. No. 4,965,188, which is incorporated herein by reference. PCR amplification of DNA involves repeated cycles of heat-denaturing the DNA, annealing two oligonucleotide primers to sequences that flank the DNA segment to be amplified, and extending the annealed primers with DNA polymerase. The primers hybridize to opposite strands of the target sequence and are oriented so that DNA synthesis by the polymerase proceeds across the region between the primers, effectively doubling the amount of the DNA segment. Moreover, because the extension products are also complementary to and capable of binding primers, each successive cycle essentially doubles the amount of DNA synthesized in the previous cycle. This results in the exponential accumulation of the specific target fragment, at a rate of approximately 2 n per cycle, where n is the number of cycles. Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences of target DNA sequences directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. 
     Isothermic amplification reactions are also known and can be used according to the methods of the invention. Examples of isothermic amplification reactions include strand displacement amplification (SDA) (Walker, et al.  Nucleic Acids Res.  20(7):1691-6 (1992); Walker  PCR Methods Appl  3(1):1-6 (1993)), transcription-mediated amplification (Phyffer, et al.,  J. Clin. Microbiol.  34:834-841 (1996); Vuorinen, et al.,  J. Clin. Microbiol.  33:1856-1859 (1995)), nucleic acid sequence-based amplification (NASBA) (Compton,  Nature  350(6313):91-2 (1991), rolling circle amplification (RCA) (Lisby,  Mol. Biotechnol.  12(1):75-99 (1999)); Hatch et al.,  Genet. Anal.  15(2):35-40 (1999)) and branched DNA signal amplification (bDNA) (see, e.g., Iqbal et al.,  Mol. Cell Probes  13(4):315-320 (1999)). Other amplification methods known to those of skill in the art include CPR (Cycling Probe Reaction), SSR (Self-Sustained Sequence Replication), SDA (Strand Displacement Amplification), QBR (Q-Beta Replicase), Re-AMP (formerly RAMP), RCR (Repair Chain Reaction), TAS (Transcription Based Amplification System), and HCS. 
     The nucleic acid amplification reaction is preferably carried out using a thermal processing instrument that heats and/or cools the reaction mixture in the vessel  18  to the temperatures needed for the amplification reaction. The thermal processing instrument can also optionally comprise one or more detection mechanisms for detecting the marker nucleic acid sequence of the sample preparation controls and the one or more target nucleic acid sequences for which the sample is being tested. A preferred thermal processing instrument with built in optical detectors for amplifying and detecting nucleic acid sequences in the vessel  18  is described in commonly assigned U.S. Pat. Nos. 6,369,893 and 6,391,541, the disclosures of which are incorporated by reference herein. There are also many other known ways to control the temperature of a reaction mixture and detect nucleic acid sequences in the reaction mixture that are suitable for the present invention. For example, other instruments for nucleic acid amplification and detection are described, e.g., in U.S. Pat. Nos. 5,958,349; 5,656,493; 5,333,675; 5,455,175; 5,589,136 and 5,935,522. 
     The detection of the marker nucleic acid sequence of the sample preparation controls and of the one or more target nucleic acid sequences for which the sample is being tested is preferably carried out using probes. The reaction vessel  18  preferably has one or more transparent or light-transmissive walls through which signals from the probe may be optically detected. Preferably hybridization probes are used to detect and quantify the nucleic acid sequences. There are many different types of assays that employ nucleic acid hybridization probes. Some of these probes generate signals with a change in the fluorescence of a fluorophore due to a change in its interaction with another molecule or moiety. Typically, the interaction is brought about by changing the distance between the fluorophore and the interacting molecule or moiety. These assays rely for signal generation on fluorescence resonance energy transfer, or “FRET.” FRET utilizes a change in fluorescence caused by a change in the distance separating a first fluorophore from an interacting resonance energy acceptor, either another fluorophore or a quencher. Combinations of a fluorophore and an interacting molecule or moiety, including quenching molecules or moieties, are known as “FRET pairs.” The mechanism of FRET-pair interaction requires that the absorption spectrum of one member of the pair overlaps the emission spectrum of the other member, the first fluorophore. If the interacting molecule or moiety is a quencher, its absorption spectrum must overlap the emission spectrum of the fluorophore. Stryer, L.,  Ann. Rev. Biochem.  1978, 47: 819-846; BIOPHYSICAL CHEMISTRY part II, Techniques for the Study of Biological Structure and Function, (C. R. Cantor and P. R. Schimmel, eds., 1980), pages 448-455, and Selvin, P. R.,  Methods in Enzymology  246: 300-335 (1995). Efficient, or a substantial degree of, FRET interaction requires that the absorption and emission spectra of the pair have a large degree of overlap. The efficiency of FRET interaction is linearly proportional to that overlap. Haugland, R. P., Yguerabide, Jr., and Stryer, L.,  Proc. Natl. Acad. Sci. USA  63: 24-30 (1969). Non-FRET probes have also been described. See, e.g., U.S. Pat. No. 6,150,097. 
     Another preferred method for detection of amplification products is the 5′ nuclease PCR assay (also referred to as the TaqMan® assay) (Holland et al.,  Proc. Natl. Acad. Sci. USA  88: 7276-7280 (1991); Lee et al.,  Nucleic Acids Res.  21: 3761-3766 (1993)). This assay detects the accumulation of a specific PCR product by hybridization and cleavage of a doubly labeled fluorogenic probe (the “TaqMan®” probe) during the amplification reaction. The fluorogenic probe consists of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye. During PCR, this probe is cleaved by the 5′-exonuclease activity of DNA polymerase if, and only if, it hybridizes to the segment being amplified. Cleavage of the probe generates an increase in the fluorescence intensity of the reporter dye. 
     Another method of detecting amplification products that relies on the use of energy transfer is the “beacon probe” method described by Tyagi and Kramer ( Nature Biotech.  14:303-309 (1996)), which is also the subject of U.S. Pat. Nos. 5,119,801 and 5,312,728. This method employs oligonucleotide hybridization probes that can form hairpin structures. On one end of the hybridization probe (either the 5′ or 3′ end), there is a donor fluorophore, and on the other end, an acceptor moiety. In the case of the Tyagi and Kramer method, this acceptor moiety is a quencher, that is, the acceptor absorbs energy released by the donor, but then does not itself fluoresce. Thus when the beacon is in the open conformation, the fluorescence of the donor fluorophore is detectable, whereas when the beacon is in hairpin (closed) conformation, the fluorescence of the donor fluorophore is quenched. When employed in PCR, the molecular beacon probe, which hybridizes to one of the strands of the PCR product, is in “open conformation,” and fluorescence is detected, while those that remain unhybridized will not fluoresce (Tyagi and Kramer,  Nature Biotechnol.  14: 303-306 (1996). As a result, the amount of fluorescence will increase as the amount of PCR product increases, and thus may be used as a measure of the progress of the PCR. 
     To be confident about the detection, or lack thereof, of a target nucleic acid sequence in a sample, one should control for the integrity of the sample preparation. This is why the sample preparation controls are subjected to the same treatment as the target entities (e.g., target cells, spores, viruses, or microorganisms containing a target nucleic acid sequence) in the sample. If the marker nucleic acid sequence of the sample preparation controls is detected, then the sample preparation is deemed satisfactory. If the presence of the marker nucleic acid sequence cannot be detected, then the sample preparation is deemed inadequate and the outcome of the test for the target nucleic acid sequence is deemed “unresolved”. Preferably, the presence or absence of the marker nucleic acid sequence is detected by determining if a signal from a hybridization probe capable of binding to the marker nucleic acid sequence exceeds a threshold level, e.g., a predetermined fluorescent threshold level that must be met or exceeded for the assay to be deemed valid. 
     To operate the valve  16  of  FIGS. 3-8 , a motor such as a stepper motor is typically coupled to the toothed periphery  29  of the disk portion  22  to rotate the valve  16  relative to the housing  12  for distributing fluid with high precision. The motor can be computer-controlled according to the desired protocol. A linear motor or the like is typically used to drive the piston  54  up and down with precision to provide accurate metering, and may also be computer-controlled according to the desired protocol. 
     The use of a single valve produces high manufacturing yields due to the presence of only one failure element. The concentration of the fluid control and processing components results in a compact apparatus (e.g., in the form of a small cartridge) and facilitates automated molding and assembly. As discussed above, the system advantageously includes dilution and mixing capability, intermediate wash capability, and positive pressurization capability. The fluid paths inside the system are normally closed to minimize contamination and facilitate containment and control of fluids within the system. The reaction vessel is conveniently detachable and replaceable, and may be disposable in some embodiments. 
     The components of the fluid control and processing system may be made of a variety of materials that are compatible with the fluids being used. Examples of suitable materials include polymeric materials such as polypropylene, polyethylene, polycarbonate, acrylic, or nylon. The various chambers, channels, ports, and the like in the system may have various shapes and sizes. 
       FIG. 10  shows another embodiment in which a piston assembly  210  including a piston rod  212  connected to a piston shaft  214  having a smaller cross-section than the rod  212  for driving small amounts of fluids. The thin piston shaft  214  may bend under an applied force if it is too long. The piston rod  212  moves along the upper portion of the barrel or housing  216 , while the piston shaft  214  moves along the lower portion of the barrel  216 . The movement of the piston rod  212  guides the movement of the piston shaft  214 , and absorbs much of the applied force so that very little bending force is transmitted to the thin piston shaft  214 . 
       FIG. 11  shows another embodiment in which the sample is pre-filtered before being mixed with the sample preparation controls. The sample is preferably pre-filtered in a side chamber  220  that is incorporated into the device. The side chamber  220  includes an inlet port  222  and an outlet port  224 . In this example, the side chamber  220  includes a filter  226  disposed at the inlet port  222 . Sample fluid is directed to flow via the inlet port  222  into the side chamber  220  and out via the outlet port  224  for side filtering. This allows filtering of a fluid sample or the like using the fluid control device of the invention. The fluid may be recirculated to achieve better filtering by the filter  226 . This prefiltering is useful to remove coarse material, that might otherwise clog up the other parts of the device, before mixing the sample with the sample preparation controls. After the sample is pre-filtered, it is mixed with the sample preparation controls, e.g., in the chamber  66  of  FIG. 9C  or another chamber of the housing  12 . The use of a side chamber is advantageous, for instance, to avoid contaminating the valve and the other chambers in the device. 
     The above-described arrangements of devices and methods are merely illustrative of applications of the principles of this invention and many other embodiments and modifications may be made without departing from the spirit and scope of the invention as defined in the claims. 
     For example, although a rotary-valve cartridge has been described as a preferred embodiment, the sample preparation control of the present invention is suitable for many other cartridge designs. Alternative cartridge designs are described in U.S. Pat. Nos. 6,391,541, 6,440,725, and 6,168,948 the disclosures of which are incorporated by reference herein. Moreover, when a rotary valve cartridge is used, the cartridge may have more or fewer chamber than shown in the preferred embodiments and many different sample preparation protocols may be executed. 
     The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.