U.S. patent application number 11/159008 was filed with the patent office on 2005-10-27 for hybrid automated continuous nucleic acid and protein analyzer using real-time pcr and liquid bead arrays.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Colston, Billy W. JR., Langlois, Richard G., Milanovich, Fred P., Nasarabadi, Shanavaz L., Skowronski, Evan W..
Application Number | 20050239192 11/159008 |
Document ID | / |
Family ID | 31891539 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050239192 |
Kind Code |
A1 |
Nasarabadi, Shanavaz L. ; et
al. |
October 27, 2005 |
Hybrid automated continuous nucleic acid and protein analyzer using
real-time PCR and liquid bead arrays
Abstract
A nucleic acid assay system for analyzing a sample using a
reagent. A sample and reagent delivery unit is operatively
connected to a thermal cycler for delivering the sample and the
reagent to the thermal cycler. A hybridization chamber is
operatively connected to the thermal cycler. A flow cytometer is
operatively connected to the hybridization chamber.
Inventors: |
Nasarabadi, Shanavaz L.;
(Livermore, CA) ; Langlois, Richard G.;
(Livermore, CA) ; Colston, Billy W. JR.; (San
Ramon, CA) ; Skowronski, Evan W.; (Odenton, MD)
; Milanovich, Fred P.; (Lafayette, CA) |
Correspondence
Address: |
Eddie E. Scott
Assistant Laboratory Counsel
Lawrence Livermore National Laboratory
P.O. Box 808, L-703
Livermore
CA
94551
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
31891539 |
Appl. No.: |
11/159008 |
Filed: |
June 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11159008 |
Jun 21, 2005 |
|
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10643797 |
Aug 19, 2003 |
|
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60406159 |
Aug 26, 2002 |
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Current U.S.
Class: |
435/287.1 |
Current CPC
Class: |
Y02A 90/26 20180101;
G01N 2001/2217 20130101; G01N 1/2202 20130101; G01N 15/1459
20130101; G01N 1/2211 20130101; Y02A 90/10 20180101; G01N 35/08
20130101 |
Class at
Publication: |
435/287.1 |
International
Class: |
C12M 001/34; C12M
003/00 |
Goverment Interests
[0002] The United States Government has rights in this invention
pursuant to Contract No. W-7405-ENG-48 between the United States
Department of Energy and the University of California for the
operation of Lawrence Livermore National Laboratory.
Claims
The invention claimed is:
1. A nucleic acid assay apparatus for analyzing a sample using a
reagent, comprising: a thermal cycler, a sample and reagent
delivery unit operatively connected to said thermal cycler for
delivering the sample and the reagent to said thermal cycler, a
hybridization chamber operatively connected to said thermal cycler,
and a flow cytometer operatively connected to said hybridization
chamber.
2. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with
each bead having a unique spectral address.
3. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize polystyrene
latex microspheres beads with each bead having a unique spectral
address.
4. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with
varying ratios of red and orange infrared dyes giving each bead a
unique spectral address.
5. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize a 100-plex
array of beads with varying ratios of red and orange infrared dyes
giving each bead a unique spectral address.
6. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with
each bead having a capture antibody specific for a target
antigen.
7. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with at
least one bead having an anthrax capture antibody specific for a
target anthrax antigen.
8. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with at
least one bead having a plague capture antibody specific for a
target plague antigen.
9. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with at
least one bead having a small pox capture antibody specific for a
target small pox antigen.
10. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with at
least one bead having a botox capture antibody specific for a
target botox antigen.
11. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with
each bead having a capture antibody specific for a target antigen
and a fluorescent reporter.
12. The nucleic acid assay apparatus of claim 1 wherein said flow
cytometer utilizes at least one laser.
13. The nucleic acid assay apparatus of claim 1 wherein said flow
cytometer utilizes a red laser.
14. The nucleic acid assay apparatus of claim 1 wherein said flow
cytometer utilizes a green laser.
15. The nucleic acid assay apparatus of claim 1 wherein said flow
cytometer utilizes a red laser and a green laser.
16. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with
each bead having a fluorescent reporter and said flow cytometer
utilizes at least one laser for bead interrogation by fluoresce of
said fluorescent reporter.
17. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with
each bead having a capture antibody specific for a target antigen
and a fluorescent reporter and said flow cytometer utilizes at
least one laser for bead interrogation.
18. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber and said flow cytometer utilize beads with
each bead having a capture antibody specific for a target antigen
and a fluorescent reporter and said flow cytometer utilizes a green
laser for bead interrogation by fluoresce of said fluorescent
reporter.
19. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber includes a heater.
20. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber includes a fan.
21. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber includes a heater and a fan.
22. The nucleic acid assay apparatus of claim 1 wherein said
hybridization chamber includes a heater, a fan, and a temperature
control sensor.
23. A nucleic acid assay apparatus for analyzing a sample using a
reagent, comprising: thermal cycler means, sample and reagent
delivery means operatively connected to said thermal cycler means
for delivering the sample and the reagent to said thermal cycler
means, hybridization means operatively connected to said thermal
cycler means, and flow cytometer means operatively connected to
said hybridization means.
24. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with each bead having a unique spectral address.
25. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize
polystyrene latex microspheres beads with each bead having a unique
spectral address.
26. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with varying ratios of red and orange infrared dyes giving each
bead a unique spectral address.
27. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize a
100-plex array of beads with varying ratios of red and orange
infrared dyes giving each bead a unique spectral address.
28. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with each bead having a capture antibody specific for a target
antigen.
29. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with at least one bead having an anthrax capture antibody specific
for a target anthrax antigen.
30. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with at least one bead having a plague capture antibody specific
for a target plague antigen.
31. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with at least one bead having a small pox capture antibody specific
for a target small pox antigen.
32. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with at least one bead having a botox capture antibody specific for
a target botox antigen.
33. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with each bead having a capture antibody specific for a target
antigen and a fluorescent reporter.
34. The nucleic acid assay apparatus of claim 23 wherein said flow
cytometer means utilizes at least one laser.
35. The nucleic acid assay apparatus of claim 23 wherein said flow
cytometer means utilizes a red laser.
36. The nucleic acid assay apparatus of claim 23 wherein said flow
cytometer means utilizes a green laser.
37. The nucleic acid assay apparatus of claim 23 wherein said flow
cytometer means utilizes a red laser and a green laser.
38. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with each bead having a fluorescent reporter and said flow
cytometer means utilizes at least one laser for bead interrogation
by fluoresce of said fluorescent reporter.
39. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with each bead having a capture antibody specific for a target
antigen and a fluorescent reporter and said flow cytometer means
utilizes at least one laser for bead interrogation.
40. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means and said flow cytometer means utilize beads
with each bead having a capture antibody specific for a target
antigen and a fluorescent reporter and said flow cytometer means
utilizes a green laser for bead interrogation by fluoresce of said
fluorescent reporter.
41. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means includes a heater.
42. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means includes a fan.
43. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means includes a heater and a fan.
44. The nucleic acid assay apparatus of claim 23 wherein said
hybridization means includes a heater, a fan, and a temperature
control sensor.
45. A nucleic acid assay method for analyzing a sample using a
reagent, comprising the steps of: providing a thermal cycler,
providing a hybridization unit, providing a flow cytometer,
transporting the sample and the reagent to said thermal cycler for
amplification, and analyzing the sample with said flow cytometer
utilize beads with each bead having a unique spectral address.
46. The nucleic acid assay method of claim 45 wherein said step of
analyzing the sample with said flow cytometer comprises utilizing
polystyrene latex microspheres beads with each bead having a unique
spectral address.
47. The nucleic acid assay method of claim 45 wherein said step of
analyzing the sample with said flow cytometer comprises utilizing
beads with varying ratios of red and orange infrared dyes giving
each bead a unique spectral address.
48. The nucleic acid assay method of claim 45 wherein said step of
analyzing the sample with said flow cytometer comprises utilizing a
100-plex array of beads with varying ratios of red and orange
infrared dyes giving each bead a unique spectral address.
49. The nucleic acid assay method of claim 45 wherein said step of
analyzing the sample with said flow cytometer comprises utilizing
beads with each bead having a capture antibody specific for a
target antigen.
50. The nucleic acid assay method of claim 45 wherein said step of
analyzing the sample with said flow cytometer comprises utilizing
beads with at least one bead having an anthrax capture antibody
specific for a target anthrax antigen.
51. The nucleic acid assay method of claim 45 wherein said step of
analyzing the sample with said flow cytometer comprises utilizing
beads with at least one bead having a plague capture antibody
specific for a target plague antigen.
52. The nucleic acid assay method of claim 45 wherein said step of
analyzing the sample with said flow cytometer comprises utilizing
beads with at least one bead having a small pox capture antibody
specific for a target small pox antigen.
53. The nucleic acid assay method of claim 45 wherein said step of
analyzing the sample with said flow cytometer comprises utilizing
beads with at least one bead having a botox capture antibody
specific for a target botox antigen.
54. The nucleic acid assay method of claim 45 wherein said step of
analyzing the sample with said flow cytometer comprises utilizing
beads with each bead having a capture antibody specific for a
target antigen and a fluorescent reporter.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of co-pending
U.S. patent application Ser. No. 10/643,797 filed Aug. 19, 2003 and
titled, "System for Autonomous Monitoring of Bioagents." U.S.
patent application Ser. No. 10/643,797 filed Aug. 19, 2003 claims
the benefit of U.S. Provisional Patent Application No. 60/406,159
filed Aug. 26, 2002. U.S. patent application Ser. No. 10/643,797
filed Aug. 19, 2003 and U.S. Provisional Patent Application No.
60/406,159 filed Aug. 26, 2002 are incorporated herein by this
reference.
BACKGROUND
[0003] 1. Field of Endeavor
[0004] The present invention relates to an assay system and more
particularly to a nucleic acid assay system.
[0005] 2. State of Technology
[0006] U.S. Pat. No. 4,022,575 for an automatic chemical analyzer
to Elo H. Hansen and Jaromir Ruzicka issued May 10, 1977 provide
the following background information, "The ever increasing demand
for numbers of analyses in clinical, agricultural, pharmaceutical
and other types of analytical control has lead to the development
of a large number of various instruments for automated analysis.
The development in this field is further being stimulated by the
additional advantages gained by automation: increased precision,
decreased cost per assay and good reliability of the automated
equipment."
[0007] U.S. Pat. No. 5,589,136 for silicon-based sleeve devices for
chemical reactions, assigned to the Regents of the University of
California, inventors: M. Allen Northrup, Raymond P. Mariella, Jr.,
Anthony V. Carrano, and Joseph W. Balch, patented Dec. 31, 1996
provides the following background information: "Current instruments
for performing chemical synthesis through thermal control and
cycling are generally very large (table-top) and inefficient, and
often they work by heating and cooling of a large thermal mass
(e.g., an aluminum block). In recent years efforts have been
directed to miniaturization of these instruments by designing and
constructing reaction chambers out of silicon and silicon-based
materials (e.g., silicon, nitride, polycrystalline silicon) that
have integrated heaters and cooling via convection through the
silicon. Microfabrication technologies are now well known and
include sputtering, electrodeposition, low-pressure vapor
deposition, photolithography, and etching. Microfabricated devices
are usually formed on crystalline substrates, such as silicon and
gallium arsenide, but may be formed on non-crystalline materials,
such as glass or certain polymers. The shapes of crystalline
devices can be precisely controlled since etched surfaces are
generally crystal planes, and crystalline materials may be bonded
by processes such as fusion at elevated temperatures, anodic
bonding, or field-assisted methods. Monolithic microfabrication
technology now enables the production of electrical, mechanical,
electromechanical, optical, chemical and thermal devices, including
pumps, valves, heaters, mixers, and detectors for microliter to
nanoliter quantities of gases, liquids, and solids. Also, optical
waveguide probes and ultrasonic flexural-wave sensors can now be
produced on a microscale. The integration of these microfabricated
devices into a single system allows for the batch production of
microscale reactor-based analytical instruments. Such integrated
microinstruments may be applied to biochemical, inorganic, or
organic chemical reactions to perform biomedical and environmental
diagnostics, as well as biotechnological processing and defection.
The operation of such integrated microinstruments is easily
automated, and since the analysis can be performed in situ,
contamination is very low. Because of the inherently small sizes of
such devices, the heating and cooling can be extremely rapid. These
devices have very low power requirement and can be powered by
batteries or by electromagnetic, capacitive, inductive or optical
coupling. The small volumes and high surface-area to volume ratios
of microfabricated reaction instruments provide a high level of
control of the parameters of a reaction. Heaters may produce
temperature cycling or ramping; while sonochemical and sonophysical
changes in conformational structures may be produced by ultrasound
transducers; and polymerizations may be generated by incident
optical radiation. Synthesis reactions, and especially synthesis
chain reactions such as the polymerase chain reaction (PCR), are
particularly well-suited for microfabrication reaction instruments.
PCR can selectively amplify a single molecule of DNA (or RNA) of an
organism by a factor of 10.sup.6 to 10.sup.9. This well-established
procedure requires the repetition of heating (denaturing) and
cooling (annealing) cycles in the presence of an original DNA
target molecule, specific DNA primers, deoxynucleotide
triphosphates, and DNA polymerase enzymes and cofactors. Each cycle
produces a doubling of the target DNA sequence, leading to an
exponential accumulation of the target sequence. The PCR procedure
involves: 1) processing of the sample to release target DNA
molecules into a crude extract; 2) addition of an aqueous solution
containing enzymes, buffers deoxyribonucleotide triphosphates
(dNTPS), and aligonucleotide primers; 3) thermal cycling of the
reaction mixture between two or three temperatures (e.g., 90.
degree.-96.degree., 72.degree., and 37.degree.-55.degree. C.); and
4) detection of amplified DNA. Intermediate steps, such as
purification of the reaction products and the incorporation of
surface-bending primers, for example, may be incorporated in the
PCR procedure. A problem with standard PCR laboratory techniques is
that the PCR reactions may be contaminated or inhibited by the
introduction of a single contaminant molecule of extraneous DNA,
such as those from previous experiments, or other contaminants,
during transfers of reagents from one vessel to another. Also, PCR
reaction volumes used in standard laboratory techniques are
typically on the order of 50 microliters. A thermal cycle typically
consists of four stages: heating a sample to a first temperature,
maintaining the sample at the first temperature, cooling the sample
to a second lower temperature, and maintaining the temperature at
that lower temperature. Typically, each of these four stages of a
thermal cycle requires about one minute, and thus to complete forty
cycles, for example, is about three hours. Thus, due to the large
volume typically used in standard laboratory procedures, the time
involved, as well as the contamination possibilities during
transfers of reagents from one vessel to another, there is clearly
a need for microinstruments capable of carrying out the PCR
procedure."
[0008] In an article titled, "U.S. Is Deploying a Monitor System
for Germ Attacks," by Judith Miller in The New York Times on Jan.
22, 2003, it was reported, "To help protect against the threat of
bioterrorism, the Bush administration on Wednesday will start
deploying a national system of environmental monitors that is
intended to tell within 24 hours whether anthrax, smallpox and
other deadly germs have been released into the air, senior
administration officials said today. The system uses advanced data
analysis that officials said had been quietly adapted since the
September 11 attacks and tested over the past nine months. It will
adapt many of the Environmental Protection Agency's 3,000 air
quality monitoring stations throughout the country to register
unusual quantities of a wide range of pathogens that cause diseases
that incapacitate and kill . . . . The new environmental
surveillance system uses monitoring technology and methods
developed in part by the Department of Energy's national
laboratories. Samples of DNA are analyzed using polymerase chain
reaction techniques, which examine the genetic signatures of the
organisms in a sample, and make rapid and accurate evaluations of
that organism . . . . Officials who helped develop the system said
that tests performed at Dugway Proving Ground in Utah and national
laboratories showed that the system would almost certainly detect
the deliberate release of several of the most dangerous pathogens.
`Obviously, the larger the release, the greater the probability
that the agent will be detected,` an official said. `But given the
coverage provided by the E.P.A. system, even a small release,
depending on which way the wind was blowing and other
meteorological conditions, is likely to be picked up.`"
[0009] In an article titled, "Biodetectors Evolving, Monitoring
U.S. Cities," by Sally Cole in the May 2003 issue of Homeland
Security Solutions, it was reported, "The anthrax letter attacks of
2001, and subsequent deaths of five people, brought home the
reality of bioterrorism to Americans and provided a wake-up call
for the U.S. government about the need for a method to detect and
mitigate the impact of any such future attacks. Long before the
anthrax letter attacks, scientists at two of the U.S. Department of
Energy's national laboratories, Lawrence Livermore National
Laboratory (LLNL) and Los Alamos National Laboratory (LANL), were
busy pioneering a "biodetector" akin to a smoke detector to rapidly
detect the criminal use of biological agents. This technology is
now expected to play a large role in the U.S. government's recently
unveiled homeland security counter-terrorism initiative, Bio-Watch,
which is designed to detect airborne bioterrorist attacks on major
U.S. cities within hours. Announced back in January, Bio-Watch is a
multi-faceted, multi-agency program that involves the U.S.
Department of Energy, the Environmental Protection Agency (EPA),
and the U.S. Department of Health and Human Services' Centers for
Disease Control and Prevention (CDC). Many of the EPA's 3,000
air-quality monitoring stations throughout the country are being
adapted with biodetectors to register unusual quantities of a wide
range of pathogens that cause diseases that incapacitate and kill,
according to the EPA. The nationwide network of environmental
monitors and biodetectors, which reportedly will eventually monitor
more than 120 U.S. cities, is expected to detect and report a
biological attack within 24 hours. Citing security reasons, the EPA
declined to disclose further details about the program at this time
. . . . The Autonomous Pathogen Detection System (APDS) is a
file-cabinet-sized machine that sucks in air, runs tests, and
reports the results itself. APDS integrates a flow cytometer and
real-time PCR detector with sample collection, sample preparation,
and fluidics to provide a compact, autonomously operating
instrument capable of simultaneously detecting multiple pathogens
and/or toxins. `The system is designed for fixed locations,` says
Langlois, `where it continuously monitors air samples and
automatically reports the presence of specific biological agents.
APDS is targeted for domestic applications in which the public is
at high risk of exposure to covert releases of bioagents--subway
systems, transportation terminals, large office complexes, and
convention centers . . . . APDS provides the ability to measure up
to 100 different agents and controls in a single sample,` Langlois
says. `It's being used in public buildings right now.` The latest
evolution of the biodetector, APDS-II, uses bead-capture
immunoassays and a compact flow cytometer for the simultaneous
identification of multiple biological simulants. Laboratory tests
have demonstrated the fully autonomous operation of APDS-II for as
long as 24 hours."
SUMMARY
[0010] Features and advantages of the present invention will become
apparent from the following description. Applicants are providing
this description, which includes drawings and examples of specific
embodiments, to give a broad representation of the invention.
Various changes and modifications within the spirit and scope of
the invention will become apparent to those skilled in the art from
this description and by practice of the invention. The scope of the
invention is not intended to be limited to the particular forms
disclosed and the invention covers all modifications, equivalents,
and alternatives falling within the spirit and scope of the
invention as defined by the claims.
[0011] The present invention provides a hybrid nucleic acid assay
system for analyzing a sample using a reagent comprising a thermal
cycler, a sample and reagent delivery unit operatively connected to
the thermal cycler for delivering the sample and the reagent to the
thermal cycler, a hybridization chamber operatively connected to
the thermal cycler, and a flow cytometer operatively connected to
the hybridization chamber. The present invention also provides a
real time nucleic acid assay method for analyzing a sample using a
reagent. The method comprises the steps of providing a thermal
cycler, providing a hybridization unit, providing a flow cytometer,
transporting the sample and the reagent to said thermal cycler for
amplification, and analyzing the sample with said flow cytometer
utilize beads with each bead having a unique spectral address.
[0012] The hybrid nucleic acid analyzer system has many uses. For
example, the hybrid nucleic acid analyzer system has use for
clinical analysis of blood bank samples in a continuous 24/7
analysis of pathogens. The hybrid nucleic acid analyzer system has
use in diagnostic labs in hospitals for toxin, protein nucleic acid
and mnage analysis in clinical samples such as blood, saliva,
urine, fecal matter, etc. The hybrid nucleic acid analyzer system
has uses as fly away lab or integrated into a continuous monitoring
of environmental samples for detection of Biothreat agents. The
hybrid nucleic acid analyzer system also has use in automated
processing, amplification and detection of biological molecules in
forensic samples. The hybrid nucleic acid analyzer system can also
be used a point detector for automated clinical testing, analysis
and archiving in event of an outbreak. The hybrid nucleic acid
analyzer system can also be used to detect proteins and toxins both
in the clinic as well as from the environment.
[0013] The invention is susceptible to modifications and
alternative forms. Specific embodiments are shown by way of
example. It is to be understood that the invention is not limited
to the particular forms disclosed. The invention covers all
modifications, equivalents, and alternatives falling within the
spirit and scope of the invention as defined by the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The accompanying drawings, which are incorporated into and
constitute a part of the specification, illustrate specific
embodiments of the invention and, together with the general
description of the invention given above, and the detailed
description of the specific embodiments, serve to explain the
principles of the invention.
[0015] FIG. 1 is a fluidic diagram of illustrates one embodiment of
a hybrid nucleic acid analyzer constructed in accordance with the
present invention.
[0016] FIG. 2 shows additional details of the reagent delivery
system of the hybrid nucleic acid analyzer system illustrated in
FIG. 1.
[0017] FIG. 3 shows additional details of the thermal cycler of the
hybrid nucleic acid analyzer system illustrated in FIG. 1.
[0018] FIG. 4 shows additional details of the hybridization chamber
of the hybrid nucleic acid analyzer system illustrated in FIG.
1.
[0019] FIG. 5 shows schematics of the hybridization chamber
illustrated in FIG. 4.
[0020] FIG. 6 shows additional details of the flow cytometer of the
hybrid nucleic acid analyzer system illustrated in FIG. 1.
[0021] FIG. 7 shows the beads used in the hybridization chamber and
the flow cytometer illustrated in FIG. 1.
[0022] FIG. 8 illustrates how the beads are used in the
hybridization chamber and the flow cytometer illustrated in FIG.
1.
[0023] FIG. 9 provides additional information illustrating how the
beads are used in the hybridization chamber and in the flow
cytometer illustrated in FIG. 1.
[0024] FIG. 10 illustrates how the beads are analyzed in the flow
cytometer shown in FIG. 6.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Referring to the drawings, to the following detailed
description, and to incorporated materials, detailed information
about the invention is provided including the description of
specific embodiments. The detailed description serves to explain
the principles of the invention. The invention is susceptible to
modifications and alternative forms. The invention is not limited
to the particular forms disclosed. The invention covers all
modifications, equivalents, and alternatives falling within the
spirit and scope of the invention as defined by the claims.
[0026] Nucleic acid amplification and detection is a widely used
technique for conducting biological research. Utilization is
applied to an increasing range of applications from diagnostics in
bench-top research to the clinical arena, to genomic screening for
drug discovery, to toxicology screening for contamination, to
identification in a high throughput or conventional analysis
capacity. Conventional sample preparation and analysis techniques
for performing nucleic acid assays are time-consuming, require
trained technicians, and lack precise repeatability. New technical
developments are needed to improve the performance of nucleic acid
amplification and detection.
[0027] Early attempts to automate analytical science turned to
robotics, but the high cost of instrumentation and excessive
complexity demanded large budgets both in terms of hardware and
research effort. With the rapid growth in genomics, and proteomics,
and high throughput screening techniques, robotics has enjoyed
resurgence. The requirement for large hardware budgets and research
resources has not changed.
[0028] Referring now to FIG. 1, a fluidic diagram of illustrates
one embodiment of a hybrid nucleic acid analyzer constructed in
accordance with the present invention. This embodiment of the
hybrid nucleic acid analyzer is designated generally by the
reference numeral 100. Nucleic acid and protein analyses are
usually done on separate analysis platforms. Nucleic acid detection
is conventionally done by either Real-time Taqman PCR detection or
an end point electrophoresis. Enzyme Linked Immuno assay (ELISA) is
the method of choice for protein/antigen analysis.
[0029] One embodiment of the hybrid nucleic acid analyzer 100
provides a system for detecting and analyzing the samples: one
based on PCR and the other based on flow cytometry. The flow
cytometry subsystem uses antibodies to identify pathogens. For the
flow-cytometry subsystem, small "capture" beads that are 5
micrometers in diameter are coated with antibodies specific to the
target pathogens. The beads are color coded according to which
antibodies they hold. Once the pathogens attach to their respective
antibodies, more antibodies--those labeled with a fluorescent
dye--are added to the mix. A labeled antibody will stick to its
respective pathogen, creating a sort of bead sandwich-antibody,
pathogen, and labeled antibody. The beads flow one by one through a
flow cytometer, which illuminates each bead in turn with a laser
beam. Any bead with labeled antibodies will fluoresce. The system
can then identify which agents are present, depending on the color
of the capture bead.
[0030] The hybrid nucleic acid analyzer system 100 comprises a
reagent delivery system 101, a thermal cycler 102, a hybridization
chamber 103, and a flow cytometer 104. The reagent delivery system
101 delivers PCR reagents to the thermal cycler 102 autonomously.
On completion of cycling in the thermal cycler 102, the reaction 5
.mu.l is moved to the hybridization chamber 103. The 5 .mu.l is
mixed with 1e6 Luminex beads/ml in the hybridization chamber 103
for hybridization. The hybridized beads are then moved to the flow
cytometer 104 for analyses.
[0031] The hybrid nucleic acid analyzer system 100 utilizes nucleic
acid amplification and detection and sample preparation and
analysis techniques and information described in currently
co-pending U.S. patent application Ser. Nos. 10/189,319 and
10/643,797, both of which are owed by the Regents of the University
of California, the assignee of this application. U.S. patent
application Ser. No. 10/189,319 for an "Automated Nucleic Acid
Assay System" was filed Jul. 2, 2002 by Billy W. Colston, Jr.,
Steve B. Brown, Shanavaz L. Nasarabadi, Phillip Belgrader, Fred
Milanovich, Graham Marshall, Don Olson, and Duane Wolcott and was
published as U.S. patent application No. 2003/0032172 on Feb. 13,
2003. U.S. patent application Ser. No. 10/643,797 for a "System for
Autonomous Monitoring of Bioagents" was filed Aug. 19, 2003 by
Richard G. Langlois, Fred Milanovich, Billy W. Colston, Jr., Steve
B. Brown, Don A. Masquelier, Ray P. Mariella, and Kodomundi
Venkateswaran and was published as U.S. patent application No.
2004/0038385 on Feb. 26, 2004. The disclosures of U.S. patent
application Ser. Nos. 10/189,319 and 10/643,797 are incorporated
herein by this reference.
[0032] A LabView interface software system controls the fluidic
handling and the operation of the thermal cycler 102. The LabView
interface software system software is integrated into a form
compatible with the Graphical User Interface (GUI) used to control
and monitor the flow cytometer 104.
[0033] Referring now to FIG. 2, additional details of the reagent
delivery system 101 of the hybrid nucleic acid analyzer system 100
are shown. The reagent delivery system 101 includes a syringe pump
200 that delivers a carrier 201 to a holding coil 202. The carrier
is available to a zone fluidics system. The zone fluidics system
provides sequential injection analysis (SIA).
[0034] Zone fluidics defines a general-purpose fluidics tool,
allowing the precise manipulation of gases, liquids and solids to
accomplish very complex analytical manipulations with relatively
simple hardware. Zone fluidics is the precisely controlled
physical, chemical, and fluid-dynamic manipulation of zones of
miscible and immiscible fluids in narrow bore conduits to
accomplish sample conditioning and chemical analysis. A zone is a
volume region within a flow conduit containing at least one unique
characteristic.
[0035] A unit operation in zone fluidics comprises of a set of
fluid handling steps intended to contribute to the transformation
of the sample into a detectable species or prepare it for
manipulation in subsequent unit operations. Examples of unit
operations include sample filtering, dilution, enrichment, medium
exchange, headspace sampling, solvent extraction, matrix
elimination, de-bubbling, amplifying, hybridizing, and reacting. In
current analytical practice many of these steps are handled
manually or in isolated pieces of equipment. Integration is scant
at best, and there is a high degree of analyst involvement. In zone
fluidics, sample and reagent zones are subjected to these unit
operations in a sequential manner being transported from one unit
operation to the next under fluidic control.
[0036] Zone fluidics provides an alternative approach whereby unit
operations are performed in narrow bore conduits and the
transportation medium, instead of being mechanical as in robotics,
is fluidic. At the heart of a zone fluidics manifold is a
multi-position selection valve. Fluids are propelled and
manipulated in the manifold by means of a bi-directional flow pump.
A holding coil between the pump and valve is used to stack zones
and mix adjacent zones through dispersion and diffusion as is
practiced in sequential injection analysis (SIA).
[0037] The ports of the multi-position valve are coupled to various
reservoirs, reactors, manifold devices, and detectors as indicated.
Narrow bore conduits comprise the flow channels and provide fluid
contact between manifold devices and components. The term fluid
refers to liquids, gases, aerosols, and suspensions. Samples in
zone fluidics are not limited to liquids. Rather, gases, and
suspensions containing solids or cells are also included. Where
solid samples are used, particles are limited to a size that
ensures no blockages.
[0038] In most cases, reagents are prepared and then coupled to the
zone fluidics manifold. The metering capability of the pump and
mixing unit operations allow for reagents and standards to be
prepared in situ. Reagents can therefore be presented to the zone
fluidics manifold in an appropriately designed cartridge as
ready-made, reagent concentrates, lyophilized, or crystalline form.
Standards can be plumbed to the multi-position valve as discrete
reservoirs providing the required range of concentrations. As for
reagents though, standards can also be prepared in situ or diluted
to cover a larger dynamic range.
[0039] In the reagent delivery system 101, a syringe pump 200
delivers a carrier 201 to a holding coil 202. The carrier is
available to a zone fluidics system. The zone fluidics system
provides in sequential injection analysis (SIA). The ports of a
multi-position valve 203 of the zone fluidics sequential injection
analysis system are coupled to air reservoir 204, negative
reservoir 205, field sample reservoir 206, reagent reservoir 207,
plug 208, waste 209, bleach reservoir 210, and bleach reservoir 211
as indicated. The zone fluidics sequential injection analysis
system has an outlet 212 that delivers PCR reagents to the thermal
cycler 102.
[0040] Referring now to FIG. 3, details of the thermal cycler 102
of the hybrid nucleic acid analyzer system 100 of the present
invention are shown. Currently available Polymerase Chain Reaction
(PCR) thermal cycling units are large cumbersome and non-portable.
Some examples of commercially available semi-portable instruments
include the iCycler manufactured by Bio-Rad, the Light cycler from
Idaho Technologies and the Smart Cycler from Cepheid Inc. Real-time
PCR works by including in a reaction mix sequence specific
oligonucleotides (primer) that can be extended at its 3' end and a
third non-extendable oligonucleotide (probe) that has two
fluorescence molecules attached to its 5' and 3' end respectively.
Thus the probe is quenched due to the Fluorescence Resonance Energy
Transfer (FRET) between the two fluorescent molecules. FRET is
dependent on the sixth power of the intermolecular separation of
the two fluorophores. In the absence of primer extension, there is
no fluorescence signal detected by the fluorimeter. The enzyme DNA
polymerase has 5'-3' exo-nuclease activity as well as 5'-3'
polymerase activity. During primer extension, the fluorophore is
cleaved from the 5' end of the probe and since the fluorophore is
no longer quenched, a signal is detected by the fluorimeter. These
instruments are designed for measuring the fluorescence released
from sequence specific probes in case of a positive identification.
At present the multiplexing of nucleic acid signatures is limited
by the number of fluorophores that can be used in the commercial
instruments, due to spectral overlap of most of these
fluorophores.
[0041] The thermal cycler 102 of the present invention can be a
unit such as that described in U.S. Pat. No. 5,589,136 issued Dec.
31, 1996 to M. Allen Northrup, Raymond P. Mariella, Jr., Anthony V.
Carrano, and Joseph W. Balch and assigned to the Regents of the
University of California or in U.S. Pat. No. 6,586,233 issued Jul.
1, 2003 to William J. Benett. James B. Richards, and Fred P.
Milanovich and assigned to the Regents of the University of
California. The disclosures of U.S. Pat. No. 5,589,136 issued Dec.
31, 1996 and U.S. Pat. No. 6,586,233 issued Jul. 1, 2003 are
incorporated herein by this reference. As show in FIG. 3, a chamber
unit 300 is fabricated of circuit board material. The system can be
constructed of materials such as circuit board fiberglass, silicon,
ceramics, metal, or glass. Advantages of using circuit board
fiberglass is the fact that it is not as thermally conductive as
the other materials and the heating is more efficiently applied to
the sample rather than being conducted to surrounding materials.
Circuit board material is readily available and the technology of
producing and working with circuit board material is highly
developed. Circuit board material provides lower cost techniques
for fabrication. Printed circuit board technology incorporates
photolithography, metal etching, numerically controlled machining,
and layering technologies to produce the desired device.
[0042] As shown in FIG. 3, the thermal cycler 102 is generally
indicated at 300. The thermal cycler 102 includes a silicon-based
sleeve as a chemical reaction chamber, generally indicated at 301,
constructed of two bonded silicon parts, and which utilizes doped
polysilicon for heating and bulk silicon for convective cooling, as
described in greater detail hereinafter. The sleeve 301 includes a
slot or opening 304 into which reaction fluid, indicated at 306,
from a conduit 305 is inserted into the reaction chamber. The
conduit 305 is constructed of plastic, for example, or other
material which is inert with respect to the reaction mixture,
thereby alleviating any potential material incompatibility issues.
The sleeve is also provided with an opening 302 in which is located
an optical window 303, made, for example, of silicon nitride,
silicon dioxide, or polymers. The silicon sleeve reaction chamber
301 includes doped polysilicon for heating and bulk silicon for
convective cooling, and combines a critical ratio of silicon and
silicon nitride to the volume of material to be heated (e.g.,
liquid) in order to provide uniform heating, yet low power
requirements.
[0043] The thermal cycler 102 can be used to rapidly and
repetitively provide controlled thermal cycles to the reaction
mixture. The thermal conductivity properties of the silicon or
similar semiconducting substrate, help speed up the thermal rise
and fall times, and allow low power operation. While silicon is
unique in its thermal properties, i.e., high thermal conductivity,
a combination of silicon, silicon nitride, silicon dioxide,
polymers and other materials would provide a combination of thermal
conductivity and insulation that would allow thermal uniformity and
low power operation.
[0044] The Sample and the nucleic acid reaction mix are introduced
into the thermal cycler 102 by the Sequential Injection Analysis
fluid handling system illustrated in FIG. 2. As the sample is
continuously driven by convection through the channels it passes
through sections of channel that are temperature controlled to be
at the upper and lower temperatures required for the PCR reaction.
This continuous flow through the PCR temperature zones effectively
thermally cycles the sample.
[0045] Referring now to FIG. 4, additional details of the
hybridization chamber 103 of the hybrid nucleic acid analyzer
system 100 are shown. In the hybridization chamber 103 the
amplified mix is hybridized to luminex beads in-line before being
sent to the Flow Cytometer 104 for analysis. A multi-position valve
400 in the hybridization chamber 103 is coupled to air reservoir
401, reporter reservoir 402, hybridization thermocycler 403,
archive reservoir 404, plug 405, waste 406, holding coil 407, beads
408, and bleach reservoir 409 as indicated. The hybridization
chamber 103 uses a simple copper coil around the reaction chamber
to heat the bead and PCR mixture to the denaturation temperature.
After denaturation, the chamber is brought to its hybridization
temperature of 55-60.degree. C. with a fan placed at the bottom of
the unit. The multi-position valve 400 delivers the beads and
sample to the bead trap 410 and to a multi-position valve 411. The
multi-position valve 411 is coupled to air reservoir 412, plug 413,
Cal beads 414, and waste 415 as indicated. The hybridization
chamber 103 has an outlet 416 that delivers the hybridized sample
to the flow cytometer 104.
[0046] Referring now to FIG. 5, schematics of the hybridization
chamber 103 of the hybrid nucleic acid Analyzer are shown. A copper
tube support 500 provides a support for the hybridization chamber
103 and its associated equipment. As shown in FIG. 5, the
hybridization chamber 103 includes a temperature control sensor
501, heatshrink insulation 502, a muffin fan 503, a foil heater
504, tubing 505, and a fan 506. The heater 504 heats the chamber to
the denaturation temperature of 95.degree. C. followed by a cooling
to the required hybridization temperature with the aid of the fans
503 and 506. The PID temperature control sensor 501 precisely
monitors the denaturation and hybridization temperatures within a
couple of degrees. The hybridization chamber 103 provides a single
step movement of hybridization reagents into the chamber.
[0047] In the hybridization chamber 103 the amplified mix is
hybridized to luminex beads in-line before being sent to the Flow
Cytometer 104 for analysis. The hybridization chamber 103 heats the
bead and PCR mixture to the denaturation chamber. After
denaturation, the chamber is brought to its hybridization
temperature of 55-60.degree. C. with the fans 503 and 506. The
hybridization chamber 103 has an outlet that delivers the
hybridized sample to the flow cytometer 104.
[0048] Referring now to FIG. 6, additional details of the flow
cytometer 104 of the hybrid nucleic acid analyzer system 100 are
shown. The flow cytometer 104 comprises a Luminex LX100 Flow
Cytometer instrument 600 with a sheath source 601 and a waste
reservoir 602. The hybridized bead array is from the hybridization
camber 103 is introduced into the Luminex Flow Cytometer instrument
600 where the beads are interrogated by two lasers, a red laser for
the internal discriminator and a green laser for the external
discriminator dyes respectively. Additional details of the flow
cytometer 600 and its operation are show in FIGS. 7, 8, 9, and
10.
[0049] The protein (toxin or antigen) assay on the liquid bead
arrays is a typical sandwich assay. The antibody specific to the
antigen or a toxin is attached to the surface of Carboxylated
polystyrene beads described above. The antigen is then hybridized
to the bead sets followed by a secondary antibody to which is
attached the secondary discriminator phycoerythrin.
[0050] In order to multiplex more than four signatures, Applicants
have designed a Luminex Bead based Array analyzer. With the liquid
arrays it is possible to multiplex over 100 different organisms.
The discrimination of the polystyrene Luminex bead array is
dependent on the precise ratio of two internal discriminator dyes,
a red and an infrared dye. The signal intensity on the surface of
the bead is dependent on the concentration of the analyte in
solution, in our case the amplified DNA of a suspect agent or an
antigen or a toxin, whichever the case may be.
[0051] Referring now to FIG. 7, the beads used in the hybridization
chamber 103 and the flow cytometer 104 are illustrated. A 100-plex
Luminex liquid array 700 is generated by intercalating varying
ratios of red and orange infrared dyes into polystyrene latex
microspheres or beads 701. The process of producing varying ratios
of red and orange infrared dyes in the beads 701 is accomplished by
increasing the amount of red dye as illustrated by the arrow 702
and increasing the amount of orange dye as illustrated by the arrow
703. This gives each optically encoded bead 700 a unique spectral
address.
[0052] Referring now to FIG. 8, additional information is provided
illustrating how the beads are used in the hybridization chamber
103 and the flow cytometer 104. The beads designated by the
reference numeral 800 are coated with capture antibodies specific
for target antigens. Each bead has an attachment site specific for
a bioagent. The upper bead has an attachment site 801 for anthrax.
The next bead has an attachment site 802 for plague. The next bead
has an attachment site 803 for small pox. The next bead has an
attachment site 804 for botox. The attachment site 801 for anthrax
attaches to the anthrax bioagent 805. The attachment site 802 for
plague attaches to the plague bioagent 806. The attachment site 803
for small pox attaches to the small pox bioagent 807. The
attachment site 804 for botox attaches to the botox bioagent 808.
After incubation with the antigens, secondary or detector
antibodies are added, followed by addition of the fluorescent
reporter, phycoerythrin 809 to complete the "antigen sandwich."
[0053] Referring now to FIG. 9, additional information is provided
illustrating how the beads are used in the hybridization chamber
103 and in the flow cytometer 104. The beads are designated by the
reference numeral 900. FIG. 9 illustrates bead hybridization with
amplified PCR product. The PCR product 901 is added to labeled
luminex bead mix and denatured. This is followed by hybridization
and addition of SA-PE 902. The hybridized beads are then detected
in the Luminex flow cytometer 104.
[0054] Referring now to FIG. 10, an illustration shows how the
beads are analyzed in the flow cytometer. The beads are designated
by the reference numeral 1000. The direction of flow is shown by
the arrow 1001. The beads 1000 are interrogated one at a time. As
illustrated, one bead 1000 is shown being interrogated. A red laser
classifies the bead 1000, identifying the bead type. Subsequently a
green laser 1002 quantifies the assay on the bead surface--only
those beads with a complete sandwich will produce a fluoresce 1003
in the green, and the signal is a function of antigen
concentration.
[0055] The structural details of various embodiments of a hybrid
nucleic acid analyzer constructed in accordance with the present
invention having been illustrated in FIGS. 1-10 and described
above, the operation of the hybrid nucleic acid analyzer will now
be considered. The hybrid nucleic acid analyzer provides an
integrated nucleic acid and protein/toxin detection system capable
of in-line analysis of a complex sample within an hour or less. The
rate limiting step is the rapidity with which the nucleic acid is
amplified, the hybridization being instantaneous. The hybrid
nucleic acid analyzer has the capability of performing continuous
nucleic acid and immunoassays in a multiplex format. The hybrid
nucleic acid analyzer is a field deployable instrument for
detection of pathogens and toxins in environmental or clinical
samples. The hybrid nucleic acid analyzer takes advantage of the
multiplexing capability of the Luminex Bead arrays complexed with
multiplexed nucleic acid and protein capability developed at the
Lawrence Livermore National Laboratory.
[0056] The hybrid nucleic acid analyzer has an integrated PCR
chamber 102, DNA Hybridization chamber 103, and Luminex LX100 flow
cytometer 104 controlled by a LabView interface software for the
fluidic handling and the operation of the PCR chamber. The software
is integrated into a form compatible with the Graphical User
Interface (GUI) used to control and monitor the Luminex LX100 flow
cytometer. Control and data analysis software routines have been
written for controlling the Luminex LX100 flow cytometry.
Provisions have been made for the addition of a sample preparation
and concentration unit as well as a bead sequestering unit in order
to facilitate deep multiplexing of the agents. A sample preparation
and concentration strategy involves the use of Silicon pillar chips
capable of handling volumes of up to 100 ml or more of the sample,
releasing the DNA from the cells through lysis and concentrating it
in a small volume for analysis, thus increasing the detection limit
many folds.
[0057] The fluidics in the instrument is self-contained in order to
minimize contamination of the surroundings and the operator. This
minimizes contamination of reagents and samples, a feature not
available in commercial units. The sample and the nucleic acid
reaction mix are introduced into the thermal cycler 102 by
Sequential Injection Analysis fluid handling system 101.
[0058] The hybridization chamber 103 uses a simple copper coil
around the reaction chamber to heat the bead and PCR mixture to the
denaturation chamber. After denaturation, the chamber is brought to
its hybridization temperature of 55-60.degree. C. with a fan placed
at the bottom of the unit. A thermocouple inserted in the housing
of the hybridization chamber as well as on the silicon sleeve of
the thermal cycling chamber is used to control the temperatures via
feed back from a PID controller. In this instrument the reagents
are pre-loaded onto the system so that there is minimal user
interface.
[0059] Once the sample is introduced into the instrument 100, the
detection is autonomously done following the sequence of events
input by the researcher. Decontamination of the fluidics system is
carried out autonomously after each amplification step. The system
including the PCR chamber 102, the hybridization chamber 103, the
tubing carrying the sample to the PCR chamber and all the tubing
and fitting downstream from there on are rinsed with 5% household
bleach which we have found sufficient to effectively remove all
traces of nucleic acids or PCR product from the housing. After
every PCR run, a negative control for the agent/agents is amplified
in order to determine the efficacy of the decontamination
process.
[0060] Manual labor is the major factor for the high cost of sample
testing. The software has the capability of stacking a series of
fluidic protocols for autonomous analysis. Thus the instrument 100
can be loaded with the reagents and the samples at the beginning of
the day and the results can be accessed from a remote location.
This cuts the cost of labor as compared to the conventional way of
doing analysis. Thus with this instrument it is possible to perform
continuous analysis of samples from a known set of reagents with
minimal intervention in effect significantly reducing the cost of
the assay.
[0061] The hybrid nucleic acid analyzer 100 provides autonomous use
of both the thermal cycler 102 and the flow cytometer 104 such that
protein analysis can be performed independent of the nucleic acid
detection. For detection of antigens or toxins, the sample is
introduced directly to appropriately labeled beads followed by
hybridization to the secondary antibody and analysis of the assay
in the flow cytometer 104. The hybrid nucleic acid analyzer 100 can
be repeatedly decontaminated in between runs with a solution of 5%
household bleach.
[0062] The nucleic acid detection is done by hybridization of the
amplified PCR product with the probes attached to the surface of
the bead sets via NHS ester linkage chemistry. The PCR product is
labeled with Biotin molecules and the hybridization of the product
to the beads is followed by streptavidin phycoerythrin addition to
the hybridized reaction mix.
[0063] The hybrid nucleic acid analyzer system 100 provides a
closed integrated rapid Real-time PCR and multiplex flow analysis
instrument for identification of multiplex pathogen and toxin
within an hour with minimal exposure to the technician. The hybrid
nucleic acid analyzer system 100 combines Real-time flow through
PCR with an inline flow cytometer to detect both nucleic acids as
well as proteins. Sequential injection analysis (SIA) fluidic
system is used to deliver the sample and reagent for in-line
mixing, analysis and archiving of samples.
[0064] The unused PCR reaction mix is moved to the waste stream.
The hybrid system is decontaminated and made ready for another
round of amplification by rinsing with a 5% solution of Household
Bleach followed by water rinse. A negative reaction with water
substituted for sample is run between sample amplifications to
ensure that the system is free of carry over PCR product.
[0065] The hybrid nucleic acid analyzer 100 has many uses. For
example, the system 100 has use for clinical analysis of blood bank
samples in a continuous 24/7 analysis of pathogens. The system 100
has use in diagnostic labs. The system 100 has use as a fly away
lab or integrated into continuous monitoring of environmental
samples for detection of Biothreat agents. The system 100 also has
use in automated processing, amplification and detection of
biological molecules in forensic samples. The system 100 can also
be used for automated clinical testing, analysis and archiving in
event of an outbreak. The system 100 can also be used to detect
proteins and toxins both in the clinic as well as from the
environment.
[0066] While the invention may be susceptible to various
modifications and alternative forms, specific embodiments have been
shown by way of example in the drawings and have been described in
detail herein. However, it should be understood that the invention
is not intended to be limited to the particular forms disclosed.
Rather, the invention is to cover all modifications, equivalents,
and alternatives falling within the spirit and scope of the
invention as defined by the following appended claims.
* * * * *