U.S. patent application number 10/440422 was filed with the patent office on 2004-02-12 for automated system for isolating, amplifying and detecting a target nucleic acid sequence.
Invention is credited to Collis, Matthew P., Fort, Thomas L., Hansen, Timothy R., Thomas, Bradley S..
Application Number | 20040029260 10/440422 |
Document ID | / |
Family ID | 29550028 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040029260 |
Kind Code |
A1 |
Hansen, Timothy R. ; et
al. |
February 12, 2004 |
Automated system for isolating, amplifying and detecting a target
nucleic acid sequence
Abstract
A system and method for preparing and testing of targeted
nucleic acids is presented. The system integrates a pipetter,
extractor, assay reader, and other components, including a
selectively compliant articulated robot arm (SCARA). This
synergistic integration of previously separate diagnostic tools
creates a system and method whereby a minimum of human intervention
is required. The resulting system provides a substantially more
accurate and precise method of isolating, amplifying and detecting
targeted nucleic acids for diagnosing diseases.
Inventors: |
Hansen, Timothy R.; (Spring
Grove, PA) ; Collis, Matthew P.; (Seven Valleys,
PA) ; Thomas, Bradley S.; (Timonium, MD) ;
Fort, Thomas L.; (Finksburg, MD) |
Correspondence
Address: |
Allan M. Kiang
BECTON DICKINSON AND COMPANY
1 BECTON DRIVE
FRANKLIN LAKES
NJ
07417-1880
US
|
Family ID: |
29550028 |
Appl. No.: |
10/440422 |
Filed: |
May 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60380859 |
May 17, 2002 |
|
|
|
Current U.S.
Class: |
435/287.2 ;
414/757; 435/6.16; 435/91.2 |
Current CPC
Class: |
G01N 35/1074 20130101;
G01N 35/026 20130101; G01N 35/1065 20130101; G01N 2035/1053
20130101; G01N 2035/0405 20130101; G01N 2035/1025 20130101; G01N
2035/00366 20130101; G01N 35/0098 20130101; Y10T 436/143333
20150115; G01N 35/0099 20130101; G01N 35/028 20130101; B01L 3/50855
20130101; B01L 2300/0829 20130101 |
Class at
Publication: |
435/287.2 ;
435/6; 435/91.2; 414/757 |
International
Class: |
C12Q 001/68; C12P
019/34; C12M 001/34; B25J 001/00 |
Claims
What is claimed is:
1. An automated system for processing a component of interest
contained in at least one sample, comprising: an extraction device,
adapted to extract said component of interest from the sample; a
detection device, adapted to detect for the presence of said
component of interest extracted by said extraction device; and a
robot, adapted to automatically transfer said extracted component
of interest from said extraction device to said detection device,
wherein, said robot includes a selectively compliant articulated
robot arm (SCARA).
2. An automated system as claimed in claim 1, wherein: said
component of interest includes a nucleic acid; said extraction
device is adapted to extract said nucleic acid from the sample;
said detection device is adapted to detect the presence of said
extracted nucleic acid; and said robot is adapted to automatically
transfer said extracted nucleic acid to said detection device.
3. An automated system as claimed in claim 1, wherein: said
extracted component of interest includes a nucleic acid; and said
detection device includes a device for amplifying said nucleic
acid.
4. An automated system as claimed in claim 3, wherein: said
amplifying device includes a heater.
5. An automated system as claimed in claim 1, wherein: said
extraction device is adapted to perform non-specific capture of
said component of interest to extract said component of interest
from the sample.
6. An automated system as claimed in claim 5, wherein said
component of interest is a nucleic acid.
7. An automated system as claimed in claim 5, wherein said
component of interest is a protein.
8. An automated system as claimed in claim 1, wherein: said robot
is further adapted to transfer fluid to and remove fluid from said
extractor to assist said extractor in extracting said component of
interest.
9. An automated system as claimed in claim 1, wherein the
extraction device and detection device are integrated as a single
unit.
10. An automated system as claimed in claim 1, further comprising:
a sample rack, adapted for receiving at least one container
containing the sample; and said robot being adapted to
automatically transfer the sample from the container to the
extraction device.
11. An automated system for extracting nucleic acid from a sample,
comprising: a selectively compliant articulated robot arm (SCARA);
and an extractor adapted to extract the nucleic acid from the
sample.
12. An automated system as claimed in claim 11, wherein said SCARA
is adapted to move the sample from a container to an extractor.
13. An automated system as claimed in claim 11, wherein: said SCARA
is further adapted to move said extracted nucleic acid from said
extractor to a detector which is adapted to detect for the presence
of said nucleic acid.
14. An automated system for extracting a target nucleic acid from a
sample, comprising: an extractor, adapted to perform non-specific
capture of the target nucleic acid, to extract the nucleic acid
from the sample without separating the target nucleic acid from
non-target nucleic acid that may exist in the sample; an amplifier,
adapted to amplify said extracted nucleic acid; and a robot,
adapted to automatically convey said extracted nucleic acid to the
amplifier.
15. An automated system as claimed in claim 14, wherein: said robot
is further adapted to transfer the sample from a container to said
extractor.
16. An automated system as claimed in claim 14, wherein: said robot
is further adapted to transfer fluid to the extractor to assist the
extractor in extracting the target nucleic acid.
17. A method for processing a component of interest contained in a
sample, comprising: operating an extraction device to extract said
component of interest from the sample; operating a detector to
detect for the presence of said component of interest extracted by
said extraction device; and automatically transferring said
extracted component of interest from said extraction device to said
detection device, wherein said automatically transferring includes
operating a selectively compliant articulated robot arm (SCARA) to
perform said transferring.
18. A method as claimed in claim 17, wherein: said component of
interest includes a nucleic acid; said extraction device operating
step extracts said nucleic acid from the sample; said detection
device operating step detects the presence of said extracted
nucleic acid; and said automatically transferring step transfers
the sample to the extraction device, and automatically transfers
said extracted nucleic acid to said detection device.
19. A method as claimed in claim 17, wherein: said extracted
component of interest includes a nucleic acid; and said detection
device operating step includes amplifying said nucleic acid.
20. A method as claimed in claim 19, wherein: said amplifying
includes heating said nucleic acid.
21. A method as claimed in claim 17, wherein: said extraction
device operating step performs non-specific capture of said
component of interest to extract said component of interest from
the sample.
22. The method as claimed in claim 21, wherein said component of
interest is a nucleic acid.
23. The method as claimed in claim 21, wherein said component of
interest is a protein.
24. A method as claimed in claim 17, further comprising:
automatically transferring fluid to said extractor to assist said
extractor in extracting said component of interest.
25. The method as claimed in claim 17, wherein the extraction
device and detection device are integrated as a single unit.
26. The method as claimed in claim 17, further comprising:
receiving at least one container containing the sample in a sample
rack; and automatically transferring the sample from the container
to the extraction device using said SCARA.
27. A method for automatically extracting nucleic acid from a
sample, comprising: operating a selectively compliant articulated
robot arm (SCARA); and extracting the nucleic acid from the sample
with an extractor.
28. The method as claimed in claim 27, wherein the step of
operating said SCARA comprises: moving the sample from a container
to the extractor.
29. A method as claimed in claim 27, further comprising: operating
said SCARA to move said extracted nucleic acid from said extractor
to a detector which is adapted to detect for the presence of said
nucleic acid.
30. A method for automatically extracting a target nucleic acid
from a sample, comprising: operating an extractor to perform
non-specific capture on the target nucleic acid to extract the
nucleic acid from the sample without separating the target nucleic
acid from non-target nucleic acid that may exist in the sample;
operating an amplifier to amplify said extracted nucleic acid; and
automatically transferring said extracted nucleic acid from said
extractor to the amplifier.
31. A method as claimed in claim 30, further comprising:
automatically transferring the sample from a container to said
extractor.
32. A method as claimed in claim 30, further comprising:
automatically transfer fluid to the extractor to assist the
extractor in extracting the target nucleic acid.
33. An automated process for isolating and amplifying a target
nucleic acid sequence that may be present in a sample, said process
comprising: automatically separating the target sequence from the
fluid sample at a separation station by performing non-specific
binding on the target sequence without the need to separate the
target sequence from any non-target sequence that may exist in the
sample; and automatically transporting the separated target
sequence from the separation station to an amplifying incubation
station.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] Related subject matter is disclosed in co-pending U.S.
provisional patent application Serial No. 60/380,859, filed May 17,
2002, the entire contents of which are incorporated by
reference.
FIELD OF THE INVENTION
[0002] The invention is related to the detection of nucleic acids.
More particularly, the invention is related to a system and method
for the isolation, amplification and detection of targeted nucleic
acids in a fully automated and integrated system that comprises a
pipetter, extractor and an assay reader, as well as many other
devices, to detect the targeted nucleic acids. Related subject
matter is disclosed in co-pending U.S. provisional patent
application Serial No. 60/380,859, filed May 17, 2002, the entire
contents of which are incorporated by reference. Related subject
matter is disclosed in co-pending U.S. provisional patent
application Serial No. 60/380,859, filed May 17, 2002, the entire
contents of which are incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] None of the references described or referred to herein are
admitted to be prior art to the claimed invention.
[0004] A variety of molecular biology methodologies, such as
nucleic acid sequencing, direct detection of particular nucleic
acids sequences by nucleic acid hybridization, and nucleic acid
sequence amplification techniques, require that the nucleic acids
(DNA or RNA) be separated from the remaining cellular components.
This process generally includes the steps of collecting the cells
in a sample tube and lysing the cells with heat and/or reagent(s)
which causes the cells to burst and release the nucleic acids (DNA
or RNA) into the solution in the tube. The tube is then placed in a
centrifuge, and the sample is spun down so that the various
components of the cells are separated into density layers within
the tube. The layer of the nucleic acids can be removed from the
sample by a pipette or any suitable instrument. The samples can
then be washed and treated with appropriate reagents, such as
fluorescein probes, so that the nucleic acids can be detected in an
apparatus such as the BDProbeTec.RTM. ET system, manufactured by
Becton Dickinson and Company and described in U.S. Pat. No.
6,043,880 to Andrews et al., the entire contents of which is
incorporated herein by reference.
[0005] Although the existing techniques for separating nucleic
acids from cell samples may be generally suitable, such methods are
typically time consuming and complex. When performed manually, the
complexity and number of processing steps associated with a nucleic
acid based assay also introduce opportunities for practitioner
error, exposure to pathogens and cross contamination between
assays. Furthermore, although the centrifuging process is generally
effective in separating the nucleic acids from the other cell
components, certain impurities having the same or similar density
as the nucleic acids can also be collected in the nucleic acid
layer, and must be removed from the cell sample with the nucleic
acids.
[0006] A technique has recently been developed which is capable of
more effectively separating nucleic acids from the remaining
components of cells. This technique involves the use of
paramagnetic particles, and is described in U.S. Pat. No. 5,973,138
to Mathew P. Collis, the entire contents of which is incorporated
herein by reference.
[0007] In this technique, paramagnetic or otherwise magnetic or
magnetizable particles are placed in an acidic solution along with
cell samples. When the cell samples are lysed to release the
nucleic acids, the nucleic acids are reversibly bound to the
particles. The particles can then be separated from the remainder
of the solution by known techniques such as centrifugation,
filtering or magnetic force. The particle to which the nucleic
acids are bound can then be removed from the solution and placed in
an appropriate buffer solution, which causes the nucleic acids to
become unbound from the particles. The particles can then be
separated from the nucleic acids by any of the techniques described
above.
[0008] Examples of systems and method for manipulating magnetic
particles are described in U.S. Pat. Nos. 3,988,240, 4,895,650,
4,936,687, 5,681,478, 5,804,067 and 5,567,326, in European Patent
Application No. EP905520A1, and in published PCT Application WO
96/09550, the entire contents of each of said documents being
incorporated herein by reference.
[0009] Techniques also exist for moving solutions between
containers, such as test tubes, sample wells, and so on. In an
automated pipetting technique, in order to properly control a
pipetter device to draw fluid from a sample container such as a
test tube, it is necessary to know the level of the sample fluid in
the tube so the pipette can be lowered to the appropriate depth. It
is also necessary to detect whether the pipette tip has been
properly connected to the pipetter device. Prior methods to detect
the level of a fluid in a container include the use of electrical
conductivity detection. This method requires the use of
electrically conductive pipette tips connected to a sensitive
amplifier which detects small changes in the electrical capacitance
of the pipette tip when it comes in contact with an ionic fluid.
Pipette tip detection in this known system is achieved by touching
the end of the conductive pipette tip to a grounded conductor.
Drawbacks of this approach include the higher cost of conductive
pipette tips, and that the method only works effectively with ionic
fluids. In other words, if the fluid is non-conductive, it will not
provide a suitable electrical path to complete the circuit between
the conductors in the pipette tip.
[0010] A system and method for the measurement of the level of
fluid in a pipette tube has been described in U.S. Pat. No.
4,780,833, issued to Atake, the contents of which are herein
incorporated by reference. Atake's system and method involves
applying suction to the liquid to be measured, maintaining liquid
in a micro-pipette tube or tubes, and providing the tubes with a
storage portion having a large inner diameter and a slender tubular
portion with a smaller diameter. A pressure gauge is included for
measuring potential head in the tube or tubes. Knowing the measured
hydraulic head in the pipette tube and the specific gravity of the
liquid, the amount of fluid contained in the pipette tube can be
ascertained.
[0011] Devices used in molecular biology methodologies can
incorporate the pipette device mentioned above, with robotics, to
provide precisely controlled movements to safely and carefully move
sample biological fluids from one container to another. Typically,
these robotic devices are capable of coupling to one or more of the
aforementioned pipette tips, and employ an air pump or other
suitable pressurization device to draw the sample biological fluid
into the pipette tips.
[0012] The advent of DNA probes, which can identify a specific
bacteria by testing for the presence of a unique bacterial DNA
sequence in the sample obtained from the patient, has greatly
increased the speed and reliability of clinical diagnostic testing.
A test for the tuberculosis mycobacterium, for example, can be
completed within a matter of hours using DNA probe technology. This
allows treatment to begin more quickly and avoids the need for long
patient isolation times. The nucleic acid sequence separating
technique and the pipetting technique described above can be used
to prepare samples to be used in conjunction with DNA probe
technology for diagnostic purposes.
[0013] In the use of DNA probes for clinical diagnostic purposes, a
nucleic acid amplification reaction is usually carried out in order
to multiply the target nucleic acid into many copies or amplicons.
Examples of nucleic acid amplification reactions include strand
displacement amplification (SDA), rolling circle amplification
(RCA), self-sustained sequence replication (3SR),
transcription-mediated amplification (TMA), nucleic
acid-sequence-based amplification (NASBA), ligase chain reaction
(LCR) and polymerase chain reaction (PCR). Methods of nucleic acid
amplification are described in the literature. For example, PCR
amplification, for instance, is described by Mullis et al. in U.S.
Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Methods in
Enzymology, 155:335-350 (1987). Examples of SDA can be found in
Walker, PCR Methods and Applications, 3:25-30 (1993), Walker et al.
in Nucleic Acids Res., 20:1691-1996 (1992) and Proc. Natl. Acad.
Sci., 89:392-396 (1991). LCR is described in U.S. Pat. Nos.
5,427,930 and 5,686,272. And different TAA formats are provided in
publications such as Burg et al. in U.S. Pat. No. 5,437,990; Kacian
et al. in U.S. Pat. Nos. 5,399,491 and 5,554,516; and Gingeras et
al. in International Application No. PCT/US87/01966 and
International Publication No. WO 88/01302, and International
Application No. PCT/US88/02108 and International Publication No. WO
88/10315.
[0014] Detection of the nucleic acid amplicons can be carried out
in several ways, all involving hybridization (binding) between the
target DNA and specific probes. Many common DNA probe detection
methods involve the use of fluorescein dyes. One detection method
is fluorescence energy transfer. In this method, a detector probe
is labeled both with a fluorescein dye that emits light when
excited by an outside source, and with a quencher which suppresses
the emission of light from the fluorescein dye in its native state.
When DNA amplicons are present, the fluorescein-labeled probe binds
to the amplicons, is extended, and allows fluorescence emission to
occur. The increase of fluorescence is taken as an indication that
the disease-causing bacterium is present in the patient sample.
[0015] Other detection methods will be apparent to those skilled in
the art. For example, a single fluorescent label may be employed on
the reporter moiety with detection of a change in fluorescence
polarization in the presence of the complement of the reporter
moiety (see U.S. Pat. No. 5,593,867). Non-fluorescent labels are
also useful. For example, the reporter moiety may be labeled with a
lipophilic dye and contain a restriction site which is cleaved in
the presence of the complement of the reporter moiety (see U.S.
Pat. No. 5,550,025). Alternatively, the reporter probe may be
radiolabeled and the products resulting from synthesis of the
complement of the reporter moiety may be resolved by
electrophoresis and visualized by autoradiography. Immunological
labels may also be employed. A reporter probe labeled with a hapten
can be detected after synthesis of the complement of the reporter
moiety by first removing unreacted reporter probe (for example by
adapter-specific capture on a solid phase) and then detecting the
hapten label on the reacted reporter probe using standard
chemiluminescent or colorimetric ELISAs. A biotin label may be
substituted for the hapten and detected using methods known in the
art. Chemiluminescent compounds which include acridiuium esters
which can be used in a hybridization protection assay (HPA) and
then detected with a luminometer (see U.S. Pat. Nos. 4,950,613 and
4,946,958).
[0016] One broad category of detection devices that can be used in
the various embodiments of the invention (more fully described in
detail below), are optical readers and scanners. Several types of
optical readers or scanners exist which are capable of exciting
fluid samples with light, and then detecting any light that is
generated by the fluid samples in response to the excitation. For
example, an X-Y plate scanning apparatus, such as the CytoFluor
Series 4000 made by PerSeptive Biosystems, is capable of scanning a
plurality of fluid samples stored in an array or plate of
microwells. The apparatus includes a scanning head for emitting
light toward a particular sample, and for detecting light generated
from that sample. The apparatus includes first and second optical
cables each having first and second ends. The first ends of the
optical cables are integrated to form a single Y-shaped
"bifurcated" cable. The scanning head includes this end of the
bifurcated optical cable. The second end of the first optical cable
of the bifurcated cable is configured to receive light from a light
emitting device, such as a lamp, and the second end of the second
cable of the bifurcated cable is configured to transmit light to a
detector, such as a photomultiplier tube.
[0017] During operation, the optical head is positioned so that the
integrated end of the bifurcated optical fiber is at a suitable
position with respect to one of the microwells. The light emitting
device is activated to transmit light through the first optical
cable of the bifurcated optical cable such that the light is
emitted out of the integrated end of the bifurcated optical cable
toward the sample well. If fluid sample fluoresces in response to
the emitted light, the light produced by the fluorescence is
received by the integrated end of the optical fiber and is
transmitted through the second optical fiber to the optical
detector. The detected light is converted by the optical detector
into an electrical signal, the magnitude of which is indicative of
the intensity of the detected light. The electrical signal is
processed by a computer to determine whether the target DNA is
present or absent in the fluid sample based on the magnitude of the
electrical signal.
[0018] Another existing type of apparatus is described in U.S. Pat.
No. 5,473,437, to Blumenfeld et al. This apparatus includes a tray
having openings for receiving bottles of fluid samples. The tray
includes a plurality of optical fibers which each have an end that
terminates at a respective opening in the tray. The tray is
connected to a wheel, and rotates in conjunction with the rotation
of the wheel. The other ends of the optical fibers are disposed
circumferentially in succession about the wheel, and a light
emitting device is configured to emit light toward the wheel so
that as the wheel rotates, the ends of the optical fibers
sequentially receive the light being emitted by the light emitting
device. That is, when the wheel rotates to a first position, a
fiber extending from the wheel to one of the openings becomes
aligned with the optical axis of the light emitting device and
thus, the emitted light will enter that fiber and be transmitted to
the opening. The apparatus further include a light detector having
an optical axis aligned with the optical axis of the emitted light.
Accordingly, if the sample in the bottle housed in the opening
fluoresces due to the excitation light, the light emitted from the
sample will transmit through the optical fiber and be detected by
the detector. The wheel then continues to rotate to positions where
the ends of the other optical fibers become aligned with the
optical axis of the light emitter and light detector, and the light
emission and detection process is repeated to sample the fluid
samples in the bottles housed in the openings associated with those
fibers.
[0019] Another type of optical testing apparatus is described in
U.S. Pat. No. 5,518,923, to Berndt et al. That apparatus includes a
plurality of light emitter/light detector devices for testing a
plurality of fluid samples. The fluid samples are contained in jars
which are placed in the openings of a disk-shaped tray. The
plurality of the light emitter/detector devices are disposed in the
radial direction of the tray. Hence, as the tray rotates, the
samples in each circular row will pass by their respective light
emitter/detector device, which will transmit light into the sample
and detect any light that is generated by the sample in response to
the emitted light. In theory, this apparatus is capable of testing
more than one sample at any given time. However, in order to
achieve this multiple sample testing ability, the system must
employ a plurality of light detectors and a plurality of light
emitters. These additional components greatly increase the cost of
the system. For example, photomultiplier tubes, which are generally
quite expensive, are often used as light detector units in devices
of this type. Hence, the cost of the unit is generally increased if
more than one photomultiplier tube is used. However, it is
desirable to use as few photomultiplier tubes as possible to
maintain a competitive price for the apparatus. However, devices
which employ a single detector (e.g., photomultiplier tube) are
incapable of testing a plurality of samples without some type of
mechanical motion for each test.
[0020] A detector apparatus is also described in U.S. Pat. No.
4,343,991, to Fujiwara et al. This apparatus employs a single light
detector and a plurality of light emitting devices to read a sample
on a sample carrier, which is a substantially transparent medium.
In this apparatus, the plurality of light emitting devices transmit
light through corresponding optical fibers. The light emitted by
the optical fibers passes through the carrier and is received by
corresponding optical fibers on the opposite side of the carrier.
The receiving fibers terminate at a single light detector and the
light emitters are operated to emit light at different times.
Hence, light from only one of the emitters passes through the
carrier at any given time and is detected by the detector, which
outputs a signal proportional to the intensity of the detected
light. Therefore, a single detector can be used to detect light
from a plurality of light emitting devices. When the light passes
through a portion of the carrier that includes a sample, the
intensity of the light is decreased because some of the light is
absorbed by the sample. The amount by which the light intensity is
reduced is proportional to the concentration of the sample material
in the sample. Because the signal output by the detector is
proportional to the intensity of the detected light, the sample
concentration can thus be determined based on the output
signal.
[0021] Although the nucleic acid separating techniques, the
pipetting techniques, and the sensory techniques discussed above
exist separately, what is lacking, is an integrated system that
synergistically combines these and other tools to create an
advanced, easy-to-operate system for the isolation, amplification
and detection of targeted nucleic acids to diagnose diseases by
manipulating fluid samples. Past approaches to automate sample
processing were limited to automating portions of the process
leaving the remaining tasks to be performed by a technician. For
example, many earlier systems employ a manual centrifugation step
that requires a technician to load sample tubes into and out of an
external centrifuge. Other systems require a technician to transfer
extraction products from a nucleic acid extraction instrument to an
amplification and/or detection instrument.
[0022] Certain attempts have been made at providing limited
automation to sample handling systems. For example, certain systems
utilize Cartesian robots for moving samples from one location to
another. As known in the art, Cartesian robots can move in X, Y and
Z direction, are able to perform straight-line insertions and are
easy to program. Cartesian robots have the most rigid robotic
structure for a given length, since the axes can be supported at
both ends. Due to their rigid structure, Cartesian robots can
manipulate relatively high loads. This enables them to be used for
pick-and-place applications, machine tool loading, and stacking
parts into bins. Cartesian robots can also be used for assembly
operations and liquid dispensing systems. Examples of such uses
occur in laboratory applications (genetic research), or any task
that is high volume and repetitive.
[0023] One disadvantage of Cartesian robots, however, is that they
require a large area of space in which to operate, or, in other
words, have a large footprint to workspace ratio. Another
disadvantage is that Cartesian robots have exposed mechanical
elements which are difficult to seal from wet, corrosive or dusty
environments, and are difficult to decontaminate.
[0024] In addition, selectively compliant articulated robot arms
(SCARA) robots have been used in the genome area to pick colonies
and transfer them from a media plate to a sample plate.
[0025] Although the systems discussed above may be useful in
certain capacities, it a need exists to have a fully automated
system for processing a component of interest, wherein such
processing includes isolating, amplifying and detecting, and the
component of interest includes a specific or non specific nucleic
acid sequence and/or protein. Significant advantages can be
realized by automating the various process steps of an assay,
including greatly reducing the risk of user-error, pathogen
exposure, contamination, and spillage, while increasing efficiency.
Automating steps of an assay will also reduce the amount of
training required for practitioners and virtually eliminate sources
of injury attributable to high-volume applications.
SUMMARY OF THE INVENTION
[0026] It is therefore a general object of the invention to provide
a novel processing system that will obviate or minimize problems of
the type previously described.
[0027] In order to achieve this and other objects of the present
invention, an automated system for processing a component of
interest contained in a sample is provided comprising a sample
rack, adapted for receiving at least one container containing the
sample, an extraction device, adapted to extract said component of
interest from the sample, a detection device, adapted to detect for
the presence of said component of interest extracted by said
extraction device, and a robot, adapted to automatically transfer
the sample to the extraction device, and to automatically transfer
said extracted component of interest from said extraction device to
said detection device.
[0028] In accordance with an embodiment of the present invention,
the centrifugation step discussed above is eliminated and instead
accomplished with the use of a magnetic particle extractor
subsystem. Utilization of non-specific nucleic acid capture
provides advantages of lower reagent cost and improved robustness
relative to more complex specific capture systems. The low cost of
the non-specific capture particles (iron oxide) allows flexibility
to increase the capture matrix and scale binding capacity without
significantly impacting cost. Additionally, the system uses an
industrial grade (SCARA) robotic arm that provides accurate,
repeatable and reliable positioning of the pipetter as opposed to
laboratory liquid handling robotic platforms that use less reliable
robotic components.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The novel features and advantages of the present invention
will best be understood by reference to the detailed description of
the specific embodiments which follows, when read in conjunction
with the accompanying drawings, in which:
[0030] FIG. 1 illustrates a known method for manually preparing
multiple specimen samples for the isolation, amplification and
detection of targeted nucleic acid sequences;
[0031] FIG. 2 illustrates a perspective view of an automated
multiple specimen preparation system for the isolation,
amplification and detection of targeted nucleic acid sequences in
accordance with an embodiment of the invention;
[0032] FIG. 3 illustrates an internal view of the fully integrated
and automated multiple specimen system for the isolation,
amplification and detection of targeted nucleic acid sequences as
shown in FIG. 2 in accordance with an embodiment of the
invention;
[0033] FIG. 4 illustrates a system block diagram of the major
components of a fully integrated and automated multiple specimen
system as shown in FIG. 2 and more fully described in reference to
FIG. 3 and related figures;
[0034] FIG. 5 illustrates a perspective view of a SCARA robotic arm
used in the integrated and automated multiple specimen system for
the isolation, amplification and detection of targeted nucleic acid
sequences in accordance with an embodiment of the invention;
[0035] FIGS. 6 and 7 illustrate different perspective views of a
six channel pipetter assembly used in the integrated and automated
multiple specimen system shown in FIG. 2 for the isolation,
amplification and detection of targeted nucleic acid sequences in
accordance with an embodiment of the invention;
[0036] FIG. 8 illustrates an example of a pipetter tip used with
the pipetter assembly shown in FIGS. 6 and 7 in the integrated and
automated multiple specimen system shown in FIG. 2 for the
isolation, amplification and detection of targeted nucleic acid
sequences in accordance with an embodiment of the invention;
[0037] FIG. 9 illustrates a perspective view of a tube rack with an
identification system for use in the integrated and automated
multiple specimen system for the isolation, amplification and
detection of targeted nucleic acid sequences as shown in FIGS. 2
and 3 in accordance with an embodiment of the invention;
[0038] FIG. 10 illustrates a perspective of a portion of the tube
rack identification station as shown in FIG. 9 for use in the
integrated and automated multiple specimen system for the
isolation, amplification and detection of targeted nucleic acid
sequences in accordance with an embodiment of the invention;
[0039] FIG. 11 illustrates an exploded perspective view showing a
portion of the tube rack identification station assembly as shown
in FIG. 9 for use in the integrated and automated multiple specimen
system as shown in FIGS. 2 and 3 for the isolation, amplification
and detection of targeted nucleic acid sequences in accordance with
an embodiment of the invention;
[0040] FIG. 12 illustrates a perspective view of a pipette tip
holder station for use in the integrated and automated multiple
specimen system for the isolation, amplification and detection of
targeted nucleic acid sequences as shown in FIGS. 2 and 3 in
accordance with an embodiment of the invention;
[0041] FIG. 13 illustrates a perspective view of a pipetter tip
holder with tip exchange station for use in the integrated and
automated multiple specimen system for the isolation, amplification
and detection of targeted nucleic acid sequences as shown in FIGS.
2 and 3 in accordance with an embodiment of the invention;
[0042] FIG. 14 illustrates a perspective view of priming heater
plates for use in the integrated and automated multiple specimen
system for the isolation, amplification and detection of targeted
nucleic acid sequences as shown in FIGS. 2 and 3 in accordance with
an embodiment of the invention;
[0043] FIG. 15 illustrates an exploded perspective view of an
extractor system for use in the integrated and automated multiple
specimen system for the isolation, amplification and detection of
targeted nucleic acid sequences as shown in FIGS. 2 and 3 in
accordance with an embodiment of the invention;
[0044] FIG. 16 illustrates a perspective view of an assay reader
stage for use in the integrated and automated multiple specimen
system for the isolation, amplification and detection of targeted
nucleic acid sequences as shown in FIGS. 2 and 3 in accordance with
an embodiment of the invention;
[0045] FIG. 17 illustrates a perspective view of multiple position
microtiter plate with wells for use in the assay ready of FIG. 16
in the integrated and automated multiple specimen system for the
isolation, amplification and detection of targeted nucleic acid
sequences as shown in FIGS. 2 and 3 in accordance with an
embodiment of the invention;
[0046] FIG. 18 illustrates a perspective view of a multiple
position microtiter plate as shown in FIG. 17;
[0047] FIG. 19 illustrates a perspective view of a portion of a
microtiter well assembly as shown in FIG. 17;
[0048] FIG. 20 illustrates a perspective view of a plate sealer
gripper tool for use in applying a sealing plate to the microtiter
plate shown in FIG. 17 in the integrated and automated multiple
specimen system for the isolation, amplification and detection of
targeted nucleic acid sequences as shown in FIGS. 2 and 3 in
accordance with an embodiment of the invention; and
[0049] FIG. 21 is a flowchart illustrating an example of steps of a
method for operating the fully integrated and automated multiple
specimen system for the isolation, amplification and detection of
targeted nucleic acid sequences as shown in FIG. 2.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0050] The various features of the preferred embodiment will now be
described with reference to the figures, in which like parts are
identified with the same reference characters.
[0051] FIG. 1 illustrates a known method for manually preparing
multiple specimen samples for the isolation, amplification and
detection of targeted nucleic acid sequences which employs the
BDProbeTec.TM. ET System that provides sensitive and specific
detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae
(GC) from clinical samples. The technology is based on homogeneous
Strand Displacement Amplification (SDA) and detection of target
DNA. Currently, samples are processed, lysed and manually pipetted
from sample tubes to priming and amplification wells. The system
200, illustrated in FIG. 2 (and discussed in detail below) has been
developed to minimize pipetting and reduce hands-on time associated
with the BDProbeTec.TM. ET CT/GC assays by automating pipetting
from sample tubes to extractor, isolating the component of interest
and then transferring the component of interest to priming wells
and from priming to amplification wells.
[0052] As discussed in more detail below, the system 200 achieves
reliable automation through the use of an industrial grade robotic
arm 524 (see FIGS. 3 and 5) which in this embodiment is a
selectively compliant articulated robot arm (SCARA) that has a mean
time between failure of 20,000 hours or 10 years of single shift
use. The pipetter assembly 522 is comprised of pipetter tips 528,
as well as other components, which have a range of 20-1100
.mu.L.+-.10% with >1 year Preventative Maintenance Interval
(PMI) or 1,000,000 cycles. Unlike other clinical instrumentation,
the system 200 utilizes no perishable tubing for fluid
movement.
[0053] Procedure for Setting up the System 200
[0054] First, power is turned on in the system 200. Second, pipette
tips are loaded on to the system 200. Third, the priming and
amplification microwells are loaded onto the system 200. Then, in
step four, samples are loaded onto an instrument deck. Sample
parameters and assay type are chosen via a touch screen in step
five, and in step 6 the system 200 is enabled to run the processing
program.
[0055] The system 200 minimizes hands-on pipetting while achieving
the same CT/GC specimen results per shift as the BDProbeTec.TM. ET
manual system.
[0056] As will now be described, the system 200 shown in FIG. 2 can
be used for the isolation, amplification and detection of
components of interest in accordance with an embodiment of the
invention. These components of interest can include the specific or
non specific capture of nucleic acids and/or proteins. FIG. 3
illustrates a block diagram of a fully integrated and automated
multiple specimen system for the isolation, amplification and
detection of targeted nucleic acid sequences in accordance with an
embodiment of the invention. The automated multiple specimen
preparation system (system) 200 shown in FIG. 3, is comprised of an
assay reader stage 502, plate seals 504, LCD touch screen 506,
keyboard drawer 508, tube rack with identification system (tube ID
rack) 510, pipette tip holder 512, input sample tube rack 514,
extractor 516, 5 position tip rack reagent trough 518, waster port
520, robotic arm 524 and priming heater plates 526 (with vacuum
tool).
[0057] There exists more than one type of extraction device that
can be used in accordance with the embodiments of the invention,
more fully described below, as one skilled in the art can
appreciate. One such extraction device is extractor 516, which is
described in detail in U.S. patent application Ser. No. 09/573,540
"System and Method for Manipulating Magnetic Particles in Fluid
Samples to Collect DNA or RNA from a Sample," T. Hansen et al., and
U.S. patent application Ser. No. 09/858,889 "System and Method for
Manipulating Magnetic Particles in Fluid Samples to Collect DNA or
RNA from a Sample," T. Hansen et al. Additionally, the pipetter
assembly 522 is more fully described in U.S. patent application
Ser. No. 10/073,207 "A System and Method for Verifying the
Integrity of the Condition and Operation of a Pipetter Device For
Manipulating Fluid Samples," T. Hansen et al.
[0058] There exists more than one type of detection device that can
be used in accordance with the embodiments of the invention, more
fully described below, as one skilled in the art can appreciate.
One such detection device is the assay reader 502 more fully
described in U.S. Pat. No. 6,043,880 "Automated Optical Reader for
Nucleic Acid Assays", J. Andrews et al. these and other types of
detection devices were described briefly in the background of the
invention. The contents of each of the above referenced U.S. patent
applications and U.S. Patents are expressly incorporated herein by
reference.
[0059] FIG. 4 illustrates a conceptual block diagram at the system
200, showing the main components of the system 200, and how samples
are processed. The dashed lines illustrate when robotic arm 524 is
used to move sample material (with pipette tips 528) and other
devices. FIG. 4 includes a micro-controller (which can also be a
"local" or "remote" PC) 564, or any other suitable controller.
Throughout the following discussion, and especially in conjunction
with the accompanying description of the method illustrated in FIG.
21, operation of system 200 is controlled by a program which can be
stored and operated locally and/or remotely. A detailed description
of such devices and method of operation is excluded, as one skilled
in the relevant and related arts can understand its operation. Such
a controller can include a display 560, printer 562,
micro-controller 564, with mouse 568 and/or keypad 566, memory 558,
I/O interface 570 and data/control bus 556.
[0060] As discussed above, the automated system 200 makes use of a
robotic arm 524 to perform all the steps required to isolate and
amplify nucleic acid from a fluid sample. Components include an
input sample tube rack 510 with sample tacking mechanism (FIGS.
9-11), an extractor subsystem used to isolate and concentrate
nucleic acid from input sample (FIG. 15), heated priming and
amplification stations (FIG. 14) used for the amplification of
isolated nucleic acid and readers which monitor amplification of
specific target analytes (FIG. 16). All steps of the process are
fully automated by the use of an industrial grade robotic arm (FIG.
5) with an attached pipetting apparatus (FIGS. 6-8, 12 and 13)
capable of transferring fluids using disposable pipette tips 528 to
prevent cross contamination of liquid samples. The pipetting
assembly 522 makes use of pressure transducers to detect the
presence of filtered pipette tips 528 on the nozzle of the pipetter
and to sense liquid levels in sample test tubes (FIGS. 9-11). A
computer program that allows run-specific input to be entered via
an integrated LCD touch screen monitor 506 controls all processing
steps.
[0061] The system 200 is fully self-contained in an enclosure with
sliding acrylic windows that protects the operator from the moving
robotic arm 524 and prevents any aerosols that may be present in
the liquid samples from escaping. Replaceable containers,
accessible from below the instrument are used to collect the
contaminated pipette tips 528 and liquid waste.
[0062] Operation of the system 200 employing the SCARA robotic arm
524 will now be discussed. as can be appreciated by one skilled in
the art, a SCARA robot has motions very much like a human body.
These devices incorporate both a shoulder and elbow joint as well
as a wrist axis and a vertical motion. SCARA robots were invented
in Japan in the early 1960's and have been used extensively in many
different industries since then.
[0063] SCARA robots are ideal for a variety of general purpose
applications which require fast, repeatable, and articulate,
point-to-point movements. Examples of their uses include
palletizing & de-palletizing, loading and unloading, and
assembly. Because of their unique "elbow" motions, SCARA robots are
also ideal for applications which require constant acceleration
through circular motions, such as dispensing and gasket-forming
in-place. SCARA robot joints are all capable of rotation and can be
thoroughly sealed and safeguarded, which is necessary should the
robot be deployed in dusty or corrosive environments. SCARA robots
are generally faster than cartesian robots and can perform multiple
motions at their joints. Robotic arm 524, illustrated in FIG. 7, is
an example of a SCARA-type robotic arm. It should be also noted
that the system 200 is not limited to the use of a SCARA, but
rather can use any other suitable type of robotic device, such as
an articulated robot, that will enable the system 200 to perform
its intended functions.
[0064] The following is a description of the method described in
FIG. 21, in which reference is made to a specific use of one of the
embodiments of the invention, which is the processing of a targeted
nucleic acid. However, as has been described above. and as one
skilled in the art can appreciate, the invention is not limited to
this specific embodiment, nor to the processing of a targeted
nucleic acid, but the invention has several different embodiments,
and can instead be used for the processing of targeted or
non-targeted nucleic acids and/or targeted or non-targeted
proteins. Referring to FIG. 21, the method for operating the system
200, begins with step 102, in which the user first loads the
disposable pipette tips 528, extractor tubes, liquid reagents,
priming and amplification microwells and plate seals. Next, an
empty sample tube rack 510 is placed into the tube rack log in
station. The operator scans the tube rack bar 510 code via the
handheld bar code wand and then scans each sample tube to be tested
and places the tube into the tube rack 510. As each tube is placed
into the tube rack 510, a membrane keypad 554 mounted below the
tube rack 510 is activated, communicating the location of the tube
to the computer. The operator continues to wand each tube and
places it into the tube rack 510 until all tubes to be processed
are loaded. At the end of this process the computer has logged the
tube rack 510 identity and the patient information and location of
each tube loaded in the tube rack 510.
[0065] The tube rack 510 is then placed into the tube processing
station 546. A stationary bar code reader located below the tube
rack 510 reads the tube rack 510 identification and relates the
tube rack 510 identification to the database of patient information
logged for that particular tube rack 510. This information is
tracked to the final stage of the process when the patient results
are printed. Next the user closes the acrylic windows and initiates
the run via the LCD touch screen 506.
[0066] In step 104, robotic arm 524 picks-up pipette tips 528 and
transfers fluid from each sample tube into a corresponding
extraction tube 548. The extraction tubes are pre-filled with
magnetic particles and lysing reagents and covered with a foil film
that the robotic arm 524 punctures when dispensing the fluid sample
into the tube. The robotic arm 524 mixes the sample to resuspend
the extraction tube components. All mixing steps can be conducted,
but not necessarily, under the influence of a degaussing field to
facilitate particle dispersion. The robotic arm 524 disposes of the
pipette tips 528 and acquires new pipette tips 528 after each
sample transfer. This process continues until all of the samples
have been transferred into their corresponding extractor tubes.
Alternatively, the sample may be loaded directly into the extractor
device. In this embodiment the sample is in a container which may
be pre-filled with the necessary reagents and/or material for
extraction.
[0067] During the next step, step 106, heaters 572 within the
extractor subsystem 516 heat the sample to a suitable temperature
that causes the release of nucleic acid from the microorganisms
contained in the biological sample. The heaters 572 are then
disabled allowing the lysed samples to cool. Alternatively, instead
of using heat, or in combination with heat, the nucleic acid may be
released from the microorganisms contained in the biological sample
by chemical means. Means of chemical extraction are described in
"Chemical Pre-treatment of Plasma for Nucleic Acid Amplification
Assays," U.S. Ser. No. 10/359,180 and "Pretreatment Method for
Extraction of Nucleic Acid from Biological Samples and Kits
Therefor", U.S. Ser. No. 10/359,179.
[0068] After cool down, the robotic arm 524, in step 108, picks up
new pipette tips 528, aspirates binding reagent, dispenses and
mixes binding reagent into the first group of extraction tubes
using a different pipette tip 528 for each sample. This process
non-specifically binds the nucleic acid onto the magnetic
particles. Next, in step 110, magnets 550 within the extractor
subsystem 516 are automatically moved into position to collect the
magnetic particles to the sides of the tubes. The robotic arm 524,
using the same pipette tips 528, aspirates the waste liquid from
each extractor tube leaving the magnetic particles with attached
total nucleic acid locked to the side of the tube (step 111). The
magnets 550 are then moved to their original position below the
tubes, thus releasing the particles from the sides of the
tubes.
[0069] In step 112 robotic arm 524 picks up new pipette tips 528,
aspirates the wash reagent, dispenses, and then mixes the wash
reagent with the magnetic particles and bound nucleic acid
material. In step 114 the magnets 550 are then moved into position
to lock the particles to the sides of the tube and the robotic arm
524, using the same pipette tips 528 removes the liquid waste wash
reagent (step 115), leaving the washed, nucleic acid bound
particles locked to the side of the tubes. The magnets 550 are then
moved to the position below the tubes. In step 116 elution buffer
is aspirated into pipette tips 528. The elution buffer is then
dispensed into, and mixed with, the magnetic particles, thereby
releasing the total nucleic acid from the magnetic particles (step
117). A volume of elution buffer which is lower relative to the
input sample volume can be utilized to effectively concentrate the
nucleic acids. In step 118 the magnets 550 are moved up to the
lock-down position and the robotic arm 524 pipettes the eluted
sample containing concentrated nucleic acid into the priming wells
(step 119). Further details of the non-specific nucleic acid
binding processes are set forth in U.S. patent applications Ser.
No. 09/858,889, referenced above.
[0070] After a 20 minute room temperature incubation period, the
priming heater plates 526 are enabled, elevating the temperature of
the priming wells to a suitable heat spike temperature, which
disrupts non-specifically hybridized oligonucleotides (step 120).
In step 121, robotic arm 524 then aspirates the appropriate volume
of sample from the priming wells and dispenses it into the
amplification/reader wells. After all of the samples have been
transferred from the priming heater plate 526 to the amplification
plate 530, the robotic arm 524 picks-up the plate seal gripper tool
544, picks-up a plate seal 504 and places the plate seal 504 on the
amplification plate 530. The sealed amplification plate 530 is
transferred into the assay reader chamber 502, which maintains as
series of temperatures required for amplification of target nucleic
acids (step 122).
[0071] In step 124, The assay reader chamber 502 moves the sealed
amplification plate 530 over the read heads 552 to detect nucleic
acid amplification products. The assay reader chamber 502
determines the test result, and provides the data via a printout.
The locations of the test results match the location of the
original sample tubes. Two sets of priming plates and two sets of
amplification/reader plates are provided to support one or two
tests per sample. Further details of the assay reader are set forth
in U.S. Pat. No. 6,043,880, referenced above.
[0072] Alternative methods of sample processing include automating
nucleic acid extraction with the use of silica membranes. In this
case, lysed sample fluids mixed with binding reagents are pipetted
into an open vessel with a silica membrane suspended in the center.
A vacuum is employed to draw the sample through the membrane
trapping the nucleic acid in the membrane and allowing the
remaining waste fluid to be discarded. Reagents are then used to
release the nucleic acid from the membrane. Issues with automating
this approach require the assembly and disassembly of a vacuum
chamber. Using automation to achieve this complex task can be
problematic especially since all parts must be airtight.
Additionally, unused sections of the device must be blocked
(usually manually with tape) to allow an even vacuum to be achieved
over the active portion of the device.
[0073] Efficient capture of total nucleic acids (including low
copies of specific targets of interest) is particularly challenging
in the more viscous, high-protein samples such as plasma.. Through
optimization of the extraction process, we have developed protocols
utilizing the system 200, which allow efficient capture of nucleic
acids in viscous samples such as plasma and expressed vaginal
swabs. Key advances included minimizing protein pre-coating of
particles by introducing the particles after plasma treatment. This
reduces competition for potential binding sites between protein and
nucleic acids and reduces aggregation of particles due to
protein-protein interactions. Minimization of particle aggregation,
in turn, facilitates more efficient particle mixing. The
implementation of a degaussing field during aspiration mixing also
enhances mixing efficiency by minimizing particle aggregation due
to residual particle magnetism. The combination of these advances
allows efficient nucleic acid extraction from viscous,
proteinaceous samples without the aid of chaotropic salts.
[0074] The present invention has been described with reference to
certain exemplary embodiments thereof. However, it will be readily
apparent to those skilled in the art that it is possible to embody
the invention in specific forms other than those of the exemplary
embodiments described above. This may be done without departing
from the spirit of the invention. The exemplary embodiments are
merely illustrative and should not be considered restrictive in any
way. The scope of the invention is defined by the appended claims
and their equivalents, rather than by the preceding
description.
* * * * *