Solid material comminution or disintegration – Processes – By operations other than force of contact with solid surface
Reexamination Certificate
2001-10-04
2004-05-25
Rosenbaum, Mark (Department: 3725)
Solid material comminution or disintegration
Processes
By operations other than force of contact with solid surface
C241S002000, C241S301000
Reexamination Certificate
active
06739531
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an apparatus and method for rapidly disrupting cells or viruses.
BACKGROUND OF THE INVENTION
The extraction of nucleic acid from cells or viruses is a necessary task for many applications in the fields of molecular biology and biomedical diagnostics. Once released from the cells, the nucleic acid may be used for genetic analysis, e.g., sequencing, pathogen identification and quantification, nucleic acid mutation analysis, genome analysis, gene expression studies, pharmacological monitoring, storing of DNA libraries for drug discovery, etc. The genetic analysis typically involves nucleic acid amplification and detection using known techniques. For example, known polynucleotide amplification reactions include polymerase chain reaction (PCR), ligase chain reaction (LCR), QB replicase amplification (QBR), self-sustained sequence replication (3 SR), strand-displacement amplification (SDA), “branched chain” DNA amplification, ligation activated transcription (LAT), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), repair chain reaction (RCR), and cycling probe reaction (CPR).
The extraction of nucleic acids from cells or viruses is generally performed by physical or chemical methods. Chemical methods typically employ lysing agents (e.g., detergents, enzymes, or strong organics) to disrupt the cells and release the nucleic acid, followed by treatment of the extract with chaotropic salts to denature any contaminating or potentially interfering proteins. Such chemical methods are described in U.S. Pat. No. 5,652,141 to Henco et al. and U.S. Pat. No. 5,856,174 to Lipshutz et al. One disadvantage to the use of harsh chemicals for disrupting cells is that the chemicals are inhibitory to subsequent amplification of the nucleic acid. In using chemical disruption methods, therefore, it is typically necessary to purify the nucleic acid released from the cells before proceeding with further analysis. Such purification steps are time consuming, expensive, and reduce the amount of nucleic acid recovered for analysis.
Physical methods for disrupting cells often do not require harsh chemicals that are inhibitory to nucleic acid amplification (e.g., PCR). These physical methods, however, also have their disadvantages. For example, one physical method for disrupting cells involves placing the cells in a solution and heating the solution to a boil to break open the cell walls. Unfortunately, the heat will often denature proteins and cause the proteins to stick to the released nucleic acid. The proteins then interfere with subsequent attempts to amplify the nucleic acid. Another physical method is freeze thawing in which the cells are repeatedly frozen and thawed until the cells walls are broken. Unfortunately, freeze thawing often fails to break open many structures, most notably certain spores and viruses that have extremely tough outer layers.
Another physical method for disrupting cells is the use of a pressure instrument. With this method, a solution of mycobacterial microorganisms is passed through a very small diameter hole under high pressure. During passage through the hole, the mycobacteria are broken open by the mechanical forces and their internal contents are spilled into solution. Such a system, however, is large, expensive and requires a cooling system to prevent excessive heat from building up and damaging the contents of the lysed cells. Moreover, the instrument needs to be cleaned and decontaminated between runs and a large containment system is required when infectious material is handled. A further disadvantage to this system is that the solution must contain only particles having substantially the same size, so that it may not be used to process many untreated clinical or biological specimens.
It is also known that cells can be lysed by subjecting the cells to ultrasonic agitation. This method is disclosed by Murphy et al. in U.S. Pat. No. 5,374,522. According to the method, solutions or suspensions of cells are placed in a container with small beads. The container is then placed in an ultrasound bath until the cells disrupt, releasing their cellular components. This method has several disadvantages. First, the distribution of ultrasonic energy in the bath is not uniform, so that a technician must locate a high energy area within the bath and place the container into that area. The non-uniform distribution of ultrasonic energy also produces inconsistent results. Second, the ultrasound bath does not focus energy into the container so that the disruption of the cells often takes several minutes to complete, a relatively long period of time when compared to the method of the present invention. Third, it is not practical to carry an ultrasound bath into the field for use in biowarfare detection, forensic analysis, or on-site testing of environmental samples.
Another method for ultrasonic lysis of cells is disclosed in U.S. Pat. No. 4,983,523 to Li. According to the method, nucleic acids are released from cells, bacteria and viruses by non-invasively sonicating a sample contained within a sample container that is brought into physical contact with the vibrating element of a sonicator tuned to resonate at a frequency of 40 kHz or greater. One major problem with contacting a wall of a sample container with the vibrating element of a sonicator is that the vibration of the sonicator against the wall is very likely to cause severe damage to the wall (generally melting or cracking of the wall) leading to contamination of the work area, a health hazard to the operator, and loss of the sample to be analyzed.
SUMMARY
The present invention overcomes the disadvantages of the prior art by providing an improved apparatus and method for disrupting cells or viruses to release the nucleic acid therefrom. The apparatus and method of the present invention provides for the rapid, non-invasive lysis of cells or viruses held in a container by applying the vibrating surface of a transducer device to a wall of the container without melting, cracking, or otherwise damaging the wall of the container.
In a preferred embodiment, the apparatus comprises a container having a chamber for holding a liquid or gel that contains the cells or viruses to be disrupted. The container includes at least one wall defining the chamber, and the wall has a surface external to the chamber for contacting the vibrating surface of a transducer device. The wall thus provides an interface between the contents of the chamber and the vibrating surface of the transducer device. The apparatus also comprises a transducer device having a vibrating surface for contacting the external surface of the wall and for vibrating at an operating frequency and amplitude sufficient to generate pressure waves or pressure pulses in the chamber. The apparatus further comprises means for coupling the surface of the transducer device to the wall with a preload force sufficient to create a stress within the wall. The natural frequency of the wall, when the wall is stressed by the preload force, is equal to the operating frequency of the transducer device or differs from the operating frequency of the transducer device by less than 50% of the operating frequency of the transducer device, and more preferably by less than 25% of the operating frequency of the transducer device.
According to another aspect, the present invention provides a method for disrupting cells or viruses. The method comprises the step of holding a liquid or gel containing the cells or viruses in the chamber of a container. The container includes at least one wall defining the chamber, and the wall has a surface external to the chamber. A transducer device is coupled to the external surface of the wall with a preload force sufficient to create a stress within the wall. The transducer device is operated at a frequency and amplitude sufficient to generate pressure waves or pressure pulses in the chamber. The natural frequency of the wall, when the wall is stressed by the preload force, is equal to t
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