U.S. patent application number 10/643797 was filed with the patent office on 2004-02-26 for system for autonomous monitoring of bioagents.
Invention is credited to Brown, Steve B., Colston, Billy W. JR., Langlois, Richard G., Mariella, Ray P., Masquelier, Don A., Milanovich, Fred P., Venkateswaran, Kodomudi.
Application Number | 20040038385 10/643797 |
Document ID | / |
Family ID | 31891539 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038385 |
Kind Code |
A1 |
Langlois, Richard G. ; et
al. |
February 26, 2004 |
System for autonomous monitoring of bioagents
Abstract
An autonomous monitoring system for monitoring for bioagents. A
collector gathers the air, water, soil, or substance being
monitored. A sample preparation means for preparing a sample is
operatively connected to the collector. A detector for detecting
the bioagents in the sample is operatively connected to the sample
preparation means. One embodiment of the present invention includes
confirmation means for confirming the bioagents in the sample.
Inventors: |
Langlois, Richard G.;
(Livermore, CA) ; Milanovich, Fred P.; (Lafayette,
CA) ; Colston, Billy W. JR.; (San Ramon, CA) ;
Brown, Steve B.; (Livermore, CA) ; Masquelier, Don
A.; (Tracy, CA) ; Mariella, Ray P.; (Danville,
CA) ; Venkateswaran, Kodomudi; (Livermore,
CA) |
Correspondence
Address: |
Eddie E. Scott
Assistant Laboratory Counsel
Lawrence Livermore National Laboratory
P.O. Box 808, L-703
Livermore
CA
94551
US
|
Family ID: |
31891539 |
Appl. No.: |
10/643797 |
Filed: |
August 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60406159 |
Aug 26, 2002 |
|
|
|
Current U.S.
Class: |
435/287.1 ;
435/287.2; 435/30; 435/309.1 |
Current CPC
Class: |
Y02A 90/10 20180101;
G01N 1/2202 20130101; Y02A 90/26 20180101; G01N 15/1459 20130101;
G01N 2001/2217 20130101; G01N 1/2211 20130101; G01N 35/08
20130101 |
Class at
Publication: |
435/287.1 ;
435/287.2; 435/30; 435/309.1 |
International
Class: |
C12M 001/34 |
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. An autonomous monitoring apparatus for monitoring air, water,
soil, or other substance for bioagents, comprising: a collector for
gathering said air, water, soil, or other substance being
monitored, said collector separating selected potential bioagent
particles from said air, water, soil, or other substance; sample
preparation means for preparing a sample of said selected potential
bioagent particles, said sample preparation means operatively
connected to said collector for preparing said sample from said
air, water, soil, or other substance gathered by said collector;
and a detector for detecting said bioagents in said sample, said
detector operatively connected to said sample preparation
means.
2. The apparatus of claim 1 wherein said collector is an aerosol
collector.
3. The apparatus of claim 1 wherein said air, water, soil, or other
substance includes other particles in addition to said potential
bioagent particles and wherein said collector includes separator
means for separating said potential bioagent particles from said
other particles.
4. The apparatus of claim 3 wherein said potential bioagent
particles are of a predetermined size range and said separator
separates said potential bioagent particles are of a predetermined
size range from said other particles.
5. The apparatus of claim 4 wherein said collector is an aerosol
collector that collects air and includes means for separating said
air into a bypass air flow that does not contain said potential
bioagent particles of a predetermined particle size range and a
product air flow that contains said potential bioagent particles of
a predetermined particle size range.
6. The apparatus of claim 5 wherein said collector includes a
wetted-wall cyclone collector that receives said product air flow
and traps and concentrates said potential bioagent particles of a
predetermined particle size range in a liquid.
7. The apparatus of claim 1 including a computer and wherein said
sample preparation means is controlled by said computer.
8. The apparatus of claim 1 wherein said sample preparation means
is a means for providing an immunoassays sample.
9. The apparatus of claim 1 wherein said sample preparation means
is a means for providing a nucleic acid assays sample.
10. The apparatus of claim 1 wherein said sample preparation means
includes means for concentrating said air, water, soil, or other
substance.
11. The apparatus of claim 1 wherein said sample preparation means
includes means for purificating said air, water, soil, or other
substance.
12. The apparatus of claim 1 wherein said sample preparation means
includes means for lysis of spores in said air, water, soil, or
other substance.
13. The apparatus of claim 1 wherein said sample preparation means
includes means for mixing said air, water, soil, or other
substance.
14. The apparatus of claim 1 wherein said sample preparation means
includes means for injecting and/or aspirating a sample, means for
adding a reagent to said sample, means for mixing said sample and
said reagent, and means for transporting said sample and said
reagent.
15. The apparatus of claim 14 wherein said means for injecting
and/or aspirating said sample comprises a sequential injection
analysis system.
16. The apparatus of claim 14 wherein said means for injecting
and/or aspirating said sample comprises a flow injection analysis
system.
17. The apparatus of claim 14 wherein said means for adding a
reagent to said sample includes an injection valve.
18. The apparatus of claim 14 wherein said means for adding a
reagent to said sample includes a multi position selection
valve.
19. The apparatus of claim 14 wherein said means for mixing said
sample and the reagent includes a super serpentine reactor.
20. The apparatus of claim 14 wherein said means for transporting
said sample and said reagent is operatively connected to said means
for mixing said sample and said reagent.
21. The apparatus of claim 1 wherein said detector is a
liquid-array based multiplex immunoassay detector.
22. The apparatus of claim 21 wherein said liquid-array based
multiplex immunoassay detector utilizes optically encoded
microbeads.
23. The apparatus of claim 22 wherein said optically encoded
microbeads are coded with antibodies.
24. The apparatus of claim 22 wherein said optically encoded
microbeads are coded with fluorescently labeled antibodies.
25. The apparatus of claim 22 wherein said optically encoded
microbeads are color coded.
26. The apparatus of claim 22 wherein said optically encoded
microbeads are color coded with color emitting dyes.
27. The apparatus of claim 22 wherein said optically encoded
microbeads are small diameter polystyrene beads.
28. The apparatus of claim 22 wherein said optically encoded
microbeads are imbedded with precise ratios of red and orange
fluorescent dyes yielding an array of beads, each with a unique
spectral address and each bead is coated with capture antibodies
specific for a given antigen.
29. The apparatus of claim 22 including a flow cytometer for
analyzing said optically encoded microbeads.
30. The apparatus of claim 29 wherein said optically encoded
microbeads are optically encoded and fluorescently-labeled
microbeads and wherein said microbeads are individually
interrogated by said flow cytometer.
31. The apparatus of claim 1 wherein said detector is a multiplex
immunoassay detector.
32. The apparatus of claim 1 wherein said detector is a multiplex
PCR detector.
33. The apparatus of claim 1 including confirmation means for
confirming said bioagents in said sample.
34. The apparatus of claim 33 wherein said confirmation means is a
multiplex immunoassay detector.
35. The apparatus of claim 33 wherein said confirmation means is a
multiplex PCR detector.
36. The apparatus of claim 33 wherein said confirmation means is a
real time PCR detector.
37. The apparatus of claim 33 wherein said confirmation means
includes means for performing PCR amplification.
38. The apparatus of claim 33 wherein said confirmation means
includes means for injecting/aspirating a sample, means for adding
PCR reagent, means for mixing sample and reagent, means for
transport to PCR reactor, means for performing PCR amplification,
means for transport of amplified sample from PCR reactor, and means
for detection of PCR amplicon.
39. The apparatus of claim 33 wherein said confirmation means
includes means for injecting/aspirating a sample, means for adding
PCR reagent, means for mixing sample and reagent, means for
transport to PCR reactor, means for performing PCR amplification,
means for transport of amplified sample from PCR reactor, means for
detection of PCR amplicon, and means for decontamination and
conditioning of all exposed conduits.
40. The apparatus of claim 1 wherein said sample preparation means
includes optically encoded microbeads and bead suspension/mixer
means for suspending said microbeads for a predetermined time
period.
41. A method of monitoring air, water, soil, or other substance for
bioagents, said air, water, soil, or other substance containing
potential bioagent particles of various sizes, comprising the steps
of: gathering said air, water, soil, or other substance containing
potential bioagent particles of various sizes; separating said
potential bioagent particles by size and collecting said potential
bioagent particles of a size range that are likely to contain said
bioagents; and detecting said bioagents in said potential bioagent
particles of a size range that are likely to contain said
bioagents.
42. The method of claim 41 wherein said step of separating said
potential bioagent particles by size and collecting said potential
bioagent particles of a size range that are likely to contain said
bioagents comprises separating said air into a bypass air flow that
does not contain said potential bioagent particles of a size range
that are likely to contain said bioagents and a product air flow
that does contain said potential bioagent particles of a size range
that are likely to contain said bioagents.
43. The method of claim 41 wherein said step of separating said
potential bioagent particles by size and collecting said potential
bioagent particles of a size range that are likely to contain said
bioagents includes the step of concentrating said potential
bioagent particles of a size range that are likely to contain said
bioagents in a liquid.
44. The method of claim 41 wherein said step of detecting said
bioagents comprises mixing optically encoded microbeads with said
potential bioagent particles and detecting said bioagents with said
optically encoded microbeads.
45. The method of claim 41 wherein said step of detecting said
bioagents comprises mixing optically encoded microbeads coded with
antibodies with said potential bioagent particles and detecting
said bioagents with said and detecting said bioagents with said
optically encoded microbeads coded with antibodies.
46. The method of claim 41 wherein said step of detecting said
bioagents comprises mixing optically encoded microbeads coded with
fluorescently labeled antibodies with said potential bioagent
particles and detecting said bioagents with said and detecting said
bioagents with said optically encoded microbeads coded with
fluorescently labeled antibodies.
47. The method of claim 41 wherein said step of detecting said
bioagents comprises mixing optically encoded microbeads color coded
with color emitting dyes with said potential bioagent particles and
detecting said bioagents with said optically encoded
microbeads.
48. The method of claim 41 wherein said step of detecting said
bioagents comprises mixing optically encoded microbeads with said
potential bioagent particles and analyzing said optically encoded
microbeads in a flow cytometer.
49. The method of claim 41 including the step of confirming said
bioagents.
50. The method of claim 41 including the step of confirming said
bioagents by adding PCR reagent to said potential bioagent
particles, performing PCR amplification on said potential bioagent
particles, and detecting PCR amplicon in said potential bioagent
particles.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/406159 filed Aug. 26, 2002 titled "System
for Autonomous Monitoring of Bioagents." U.S. Provisional Patent
Application No. 60/406159 filed Aug. 26, 2002 titled "System for
Autonomous Monitoring of Bioagents" is incorporated herein by this
reference.
BACKGROUND
[0003] 1. Field of Endeavor
[0004] The present invention relates to bioagents and more
particularly to monitoring bioagents.
[0005] 2. State of Technology
[0006] There exists a critical need to develop distributed
biothreat agent sensor networks that can operate in civilian
applications. To operate in "Detect to Protect/Warn" type detection
architectures, these platforms need to have several key properties.
They need to be capable of detecting pathogens within a 1-2 hour
time window, allowing for enough time to respond to an event. They
need to be extremely low cost to maintain, since continuous
monitoring is essential for many applications. These platforms need
to have sufficient sensitivity to cover a broad geographical area
(limiting the necessary number of sensors) and have sufficient
selectivity to virtually eliminate false positives. Currently
available bio-weapons detection systems are designed primarily for
military use on the battlefield. These systems are often expensive
to deploy and ultimately unsuited for civilian protection.
[0007] 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.`"
[0008] 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
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
[0009] 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.
[0010] The present invention provides a system for monitoring air
for bioagents. Particles in the air are separated by size and the
particles of a size range that are likely to contain the bioagents
are collected. Any bioagents in the collected particles are
detected by a detector system. One embodiment of the present
invention includes confirming the bioagents by adding a PCR reagent
to the particles, performing PCR amplification on the particles,
and detecting PCR amplicon.
[0011] One embodiment of the present invention provides an
autonomous bioagent monitoring apparatus for monitoring air, water,
soil, or other substance for bioagents. A collector gathers the
air, water, soil, or other substance being monitored. A sample
preparation means for preparing a sample is operatively connected
to the collector. A detector for detecting the bioagents in the
sample is operatively connected to the sample preparation means.
One embodiment of the present invention includes confirmation means
for confirming the bioagents in the sample.
[0012] In one embodiment, the present invention provides an
autonomous monitoring apparatus for monitoring air, water, soil, or
other substance for bioagents. A collector gatherings the air,
water, soil, or other substance being monitored. The collector
separates selected potential bioagent particles from the air,
water, soil, or other substance. Sample preparation means prepares
a sample of the selected potential bioagent particles. The sample
preparation means is operatively connected to the collector for
preparing the sample from the air, water, soil, or other substance
gathered by the collector. A detector detects the bioagents in the
sample. The detector is operatively connected to the sample
preparation means.
[0013] In one embodiment the collector includes a wetted-wall
cyclone collector that receives product air flow and traps and
concentrates potential bioagent particles of a predetermined
particle size range in a liquid. In one embodiment the sample
preparation means includes means for injecting and/or aspirating a
sample, means for adding a reagent to the sample, means for mixing
the sample and the reagent, and means for transporting the sample
and the reagent. In one embodiment microbeads are optically encoded
and the optically encoded microbeads are interrogated by a laser in
detecting bioagents in the sample.
[0014] 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
[0015] 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.
[0016] FIG. 1 is a block diagram illustrating an embodiment of an
autonomous pathogen detection system constructed in accordance with
the present invention.
[0017] FIG. 2 is a block diagram illustrating another embodiment of
an autonomous pathogen detection system constructed in accordance
with the present invention.
[0018] FIG. 3 is a block diagram illustrating a specific embodiment
of the invention designated as an AUTONOMOUS PATHOGEN DETECTION
SYSTEM (APDS).
[0019] FIG. 4 is an illustration that shows the aerosol collection
system.
[0020] FIG. 5 is an illustration that shows the cap section
limiting the larger particulate size range entering the
collector.
[0021] FIG. 6 is an illustration that shows the virtual impactor
section.
[0022] FIG. 7 shows the multistage, wetted-wall cyclone collector
section.
[0023] FIGS. 8A, 8B, and 8C show details of a specific embodiment
of the aerosol collection system.
[0024] FIG. 9 is an illustration that shows another embodiment of
the aerosol collection system.
[0025] FIG. 10 illustrates a system for sample preparation and
detection.
[0026] FIGS. 11, 12, and 13 illustrate the liquid-array based
multiplex immunoassay detection system.
[0027] FIG. 14 is a block diagram illustrating the multiples
amplification and detection system.
[0028] FIG. 15 illustrates one specific embodiment of the in-line
nucleic acid amplification and detection system.
[0029] FIG. 16 is a block diagram illustrating another embodiment
of an autonomous pathogen detection system constructed in
accordance with the present invention.
[0030] FIG. 17 is a block diagram illustrating another embodiment
of an autonomous pathogen detection system constructed in
accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Referring now to the drawings, to the following detailed
description, and to incorporated materials, detailed information
about the present invention is provided including the description
of specific embodiments. The detailed description and the specific
embodiments serve 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.
[0032] Terrorists sending anthrax-contaminated packages. Militant
organizations obtaining potassium cyanide. Religious cult members
poisoning local residents to fix an election. Sadly, these
scenarios are not the plots of the three latest bestsellers, but
rather, very real incidents with a very real danger. By the
mid-1990s, the U.S. Congress began to assess the vulnerability of
the U.S. civilian population to biological terrorism and found us
considerably lacking in our ability to cope with even a small-scale
biological event. Initial thinking was that Department of Defense
technology could be readily transferred to the civilian arena.
However, upon further reflection, it was concluded that although
there was overlap between military and civilian defense needs, in
the case of a biological threat, there are marked differences: (1)
the soldier is trained and equipped with protective gear so he may
respond to a threat quickly enough to prevent a lethal dose; (2)
military intelligence usually reduces the potential threat to a
relatively small number of biological agents; and, (3) military
battlefield tactics are designed to minimize the density of
soldiers. The civilian population, however, is neither trained nor
equipped, is vulnerable to any conceivable pathogen and often
gathers in large crowds (special events, sporting venues, etc.)
where a small release could potentially infect thousands. In
response to these differences, federal agencies, including
Department of Energy, have recently begun funding directed research
efforts to reduce civilian biological terrorist
vulnerabilities.
[0033] At present there are more than 30 pathogens and toxins on
various agency threat lists. Public health personnel rarely see
most, of the pathogens so they have difficulty identifying them
quickly. In addition, many pathogenic infections aren't immediately
symptomatic, with delays as long as several days, limiting options
to control the disease and treat the patients. The lack of a
practical monitoring network capable of rapidly detecting and
identifying multiple pathogens or toxins on current threat lists
translates into a major deficiency in the United States ability to
counter biological terrorism.
[0034] Referring now to FIG. 1, an embodiment of an autonomous
pathogen detection system constructed in accordance with the
present invention is illustrated by a block diagram. The autonomous
pathogen detection system is designated generally by the reference
numeral 100. The autonomous pathogen detection system 100 provides
collection 100, sample preparation 103, and detection 105. The
collection 101 includes gathering air, water, soil or other
substance to provide an air sample, water sample, soil sample or a
sample of other substances.
[0035] After the collection 100, the sample is transferred as shown
by arrow 102 for sample preparation 103. The sample preparation 103
provides an automated sample, an immunoassays sample, and/or a
nucleic acid assays sample. In sample preparation 103 the sample
may be concentrated, purified, lisis of spores, mixed, and/or
amplified.
[0036] After sample preparation 103, the sample is transferred as
shown by arrow 104 for detection. In one embodiment of the
autonomous pathogen detection system 100, the detection is by a
multiplex immunoassay detector. In another embodiment of the
autonomous pathogen detection system 100, the detection is by a
multiplex PCR detector.
[0037] The autonomous pathogen detection system 100 provides an
apparatus and method for monitoring air, water, soil, or other
substance for particles containing bioagents. The autonomous
pathogen detection system 100 comprises a collector for gathering
the air, water, soil, or other substance being monitored; sample
preparation means for preparing a sample from the air, water, soil,
or other substance gathered by the collector; and a detector for
detecting any bioagents in the sample. In one embodiment the
collector is an aerosol collector. In other embodiments the
collector gathers water, soil, or other substances. The collector
in one embodiment includes separator means for separating the
particles of interest from other particles. The particles of
interest are of a predetermined size range.
[0038] In one embodiment the collector is an aerosol collector that
collects air and includes means for separating the air into a
bypass air flow that does not contain the particles of a
predetermined particle size range and a product air flow that does
contain the sample particles of a predetermined particle size
range. A wetted-wall cyclone collector receives the product air
flow and traps and concentrates the particles of a predetermined
particle size range in a liquid.
[0039] In one embodiment the sample preparation means is automated.
In one embodiment the sample preparation means provides an
immunoassays sample. In anther embodiment the sample preparation
means provides a nucleic acid assays sample. In another embodiment
the sample preparation means provides the sample preparation means
includes concentration of the air, water, soil, or other substance.
In anther embodiment the sample preparation means provides the
sample preparation means includes purification of the air, water,
soil, or other substance. In anther embodiment the sample
preparation means provides the sample preparation means includes
lysis of the air, water, soil, or other substance. In anther
embodiment the sample preparation means provides includes mixing of
the air, water, soil, or other substance. In anther embodiment the
sample preparation means provides includes amplification.
[0040] In one embodiment of the autonomous pathogen detection
system 100, the detector is a multiplex immunoassay detector. In
one embodiment of the autonomous pathogen detection system 100, the
detector is a multiplex PCR detector.
[0041] The primary focus of the autonomous pathogen detection
system 100 is the protection of civilians from terrorist attacks,
however, the system also has a role in protecting military
personnel from biological warfare attacks. The autonomous pathogen
detection system 100 also has uses in medical facilities and
research and development facilities. The autonomous pathogen
detection system 100 has uses in medical monitoring. There are a
variety of medical applications where monitoring for biological
pathogens would be useful. A good example of this is monitoring in
hospitals and clinics for highly infectious agents such as
tuberculosis or nosocomial diseases that can threaten the well
being of patients and health care professionals. The autonomous
pathogen detection system 100 also has uses in environmental
monitoring, that is, any application that would benefit from
environmental monitoring of biological species. One example is
continuous aerosol monitoring of bacterial and other pathogens that
could affect the health of livestock (such as the recent hoof and
mouth disease outbreak). Another example is continuous aerosol
monitoring of viruses that could affect the health of large
portions of the population (such as the recent SARS outbreak).
[0042] Referring now to FIG. 2, another embodiment of an autonomous
pathogen detection system constructed in accordance with the
present invention is illustrated by a block diagram. This
embodiment of the autonomous pathogen detection system is
designated generally by the reference numeral 200. The autonomous
pathogen detection system 200 provides collection 200, sample
preparation 203, detection 205, and confirmation 207. The
collection 201 includes gathering air, water, soil or other
substance to provide an air sample, water sample, soil sample or a
sample of other substances.
[0043] After the collection 200, the sample is transferred as
illustrated by arrow 202 for sample preparation 203. The sample
preparation 203 provides an automated sample, an immunoassays
sample, and/or a nucleic acid assays sample. In the sample
preparation 203 the sample may be concentrated, purified, lisis of
spores, mixed, and/or amplified.
[0044] After sample preparation 203, the sample is transferred as
illustrated by arrow 204 for detection. In one embodiment of the
autonomous pathogen detection system 200, the detection is by a
multiplex immunoassay detector. In another embodiment of the
autonomous pathogen detection system 200, the detection is by a
multiplex PCR detector.
[0045] After sample preparation 203 and detection 205 when a
pathogen has been detected, a sample is transferred from sample
preparation 203 to the confirmation module 207. This is illustrated
by arrow 206 in FIG. 2. In one embodiment, the system for
confirmation of a bioagent in the sample is a multiplex immunoassay
detector. In one embodiment of the autonomous pathogen detection
system 200, the system for confirmation of a bioagent in the sample
is a multiplex PCR detector. In one embodiment of the autonomous
pathogen detection system 200, the system for confirmation of a
bioagent in the sample is a real time PCR detector.
[0046] The autonomous pathogen detection system 200 provides an
apparatus and method for monitoring air, water, soil, or other
substance for particles containing bioagents. The autonomous
pathogen detection system 200 comprises a collector for gathering
the air, water, soil, or other substance being monitored; sample
preparation means for preparing a sample from the air, water, soil,
or other substance gathered by the collector; a detector for
detecting a bioagents in the sample; and a system for confirmation
of a bioagent in the sample. In one embodiment the collector is an
aerosol collector. In other embodiments the collector gathers
water, soil, or other substances. The collector in one embodiment
includes separator means for separating the particles of interest
from other particles. The particles of interest are of a
predetermined size range.
[0047] In one embodiment the collector is an aerosol collector that
collects air and includes means for separating the air into a
bypass air flow that does not contain the particles of a
predetermined particle size range and a product air flow that does
contain the sample particles of a predetermined particle size
range. A wetted-wall cyclone collector receives the product air
flow and traps and concentrates the particles of a predetermined
particle size range in a liquid.
[0048] In one embodiment the sample preparation means is automated.
In one embodiment the sample preparation means provides an
immunoassays sample. In anther embodiment the sample preparation
means provides a nucleic acid assays sample. In anther embodiment
the sample preparation means provides the sample preparation means
includes concentration of the air, water, soil, or other substance.
In anther embodiment the sample preparation means provides the
sample preparation means includes purification of the air, water,
soil, or other substance. In anther embodiment the sample
preparation means provides the sample preparation means includes
lysis of the air, water, soil, or other substance. In anther
embodiment the sample preparation means provides includes mixing of
the air, water, soil, or other substance. In anther embodiment the
sample preparation means provides includes amplification of the
sample.
[0049] In one embodiment of the autonomous pathogen detection
system 200, the detector 205 is a multiplex immunoassay detector.
In one embodiment of the autonomous pathogen detection system 200,
the detector 205 is a multiplex PCR detector.
[0050] In one embodiment of the autonomous pathogen detection
system 200, the system 207 for confirmation of a bioagent in the
sample is a multiplex immunoassay detector. In one embodiment of
the autonomous pathogen detection system 200, the system 207 for
confirmation of a bioagent in the sample is a multiplex PCR
detector. In one embodiment of the autonomous pathogen detection
system 200, the system 207 for confirmation of a bioagent in the
sample is a real time PCR detector.
[0051] Referring now to FIG. 3 through FIG. 12 a specific
embodiment of the invention designated as an AUTONOMOUS PATHOGEN
DETECTION SYSTEM (APDS) is shown. The APDS is designated generally
by the reference numeral 300. The APDS 300 integrates a flow
cytometer and 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 APDS 300 is designed for locations
where it continuously monitors air samples and automatically
reports the presence of specific biological agents. Plague and
anthrax are two of the pathogens the APDS 300 identifies, along
with a host of others. The APDS 300 includes the potential to
measure up to 100 different agents and controls in a single
sample.
[0052] The APDS 300 provides a stand-alone pathogen detection
system capable of rapid, continuous, low cost environmental
monitoring of multiple airborne biological threat agents. The
system 300 provides a "Detect to Protect/Warn" system with a number
of key properties. The system 300 is capable of detecting pathogens
within a 1-2 hour time window, allowing for enough time to respond
to an event. The system 300 is extremely low cost to maintain,
since continuous monitoring is essential for many applications. The
system 300 has sufficient sensitivity to cover a broad geographical
area (limiting the necessary number of sensors) and has sufficient
selectivity to virtually eliminate false positives.
[0053] Multiplexed assays are used to reduce reagent costs, making
long term monitoring operations possible, for example in U.S.
Postal Service mail screening. A orthogonal detection section
combines antibody-based and nucleic acid-based assays and reduces
false positives to a very low level. Antibody assays allow the
detector to respond to all types of bioagents, including those
without nucleic acids such as protein toxins. Nucleic acid assays
allow much more sensitive detection, reducing the number of sensors
needed to protect a given area. The fully autonomous aerosol
collection and sample preparation capabilities limit maintenance
requirements and makes integration into a central security or
monitoring network possible.
[0054] Referring again to FIG. 3, a block diagram illustrates the
APDS 300. In operation, an aerosol collector system continuously
samples the air and traps particles in a swirling buffer solution.
Particles of a given size distribution are selected by varying the
flow rate across a virtual impactor unit. The in-line sample
preparation system provides all sample preparation steps (i.e.,
mix, wash, incubation, etc.), and performs multiplex detection
using a Luminex flow cytometer.
[0055] In the "detection" sub-system, a collected sample is mixed
with optically encoded microbeads. Each color of microbead contains
a capture assay that is specific for a given bioagent. Fluorescent
labels are added to identify the presence of each agent on the
bound bead. Each optically encoded and fluorescently labeled
microbead is individually read in a flow cytometer, and fluorescent
intensities are then correlated with bioagent concentrations.
[0056] In the "confirmation" sub-system, PCR (nucleic acid)
amplification and detection confirms the presence of the bioagent.
An archived sample is mixed with the Taqman reagent, and then
introduced by a SIA system into a flow through polymerase chain
reaction (PCR) system. Specific nucleic acid signatures associated
with the targeted bioagent are amplified and detected using
fluorescence generated from nucleic acid replication from the
Taqman probes.
[0057] In the "Integrated Remote Control and Feedback" sub-system,
a central computer uses a simple serial based Labview control
system to control all instrument functions. A software system
provides data acquisition, real time data analysis, and result
reporting via a graphical user interface.
[0058] The APDS 300 is integrated into a self-contained "ATM" style
chassis. All fluids and reagents are contained in the instrument.
The ADPS 300 includes the following subsystems: Aerosol Collection
301, In-Line Sample Preparation 302, Detection--Liquid-Array Based
Multiplex Immunoassay Detection and/or Nucleic Assays Detection
303, Confirmation--In-Line Nucleic Acid Amplification and Detection
304, and Integrated Remote Control and Feedback 305. The subsystem
will be described in greater detail.
[0059] APDS Aerosol Collection--301
[0060] The first stage of the APADS 300 is "aerosol collection"
that provides collection of airborne particles that could contain
targeted bioagents. Aerosol release of bioagents is considered one
of the possible scenarios of a terrorist organization. One of the
methods of rapidly exposing a large population to a biowarfare
agent is through use of an aerosol (witness the effect of the
recent, relatively small-scale anthrax mailroom releases). The
aerosol collection system 301 continuously samples the air and
traps particles in a swirling buffer solution. Particles of a given
size distribution are selected by varying the flow rate across a
virtual impactor unit.
[0061] The aerosol collection system 301 is a multi-stage aerosol
collector that utilizes a low pass aerosol section and a virtual
impactor preconcentration that delivers the particles of interest
to a wetted wall cyclone collector. The virtual impactor captures
particles 1-10 gm which is the size of particles most likely to be
captured in the human lung. In the wetted wall cyclone collector,
the particles are collected in a fluid, making downstream
processing much easier. The fans and inputs to the obtain high
collection rates, up to 3000 liters of air per minute flow through
the detection system, allowing many particles to be collected over
a short period. The aerosol collection system provides improved
sensitivity and reduced collection times. An on board computer
controls air flow rates and the size range of particles collected.
A particle counter provides reaptime feedback on the size and
quantity of particles collected.
[0062] As shown by FIGS. 4 and 5, a very high volume flow of
aerosol particles is drawn into an annular slot 401 formed in a cap
402 that is designed to only allow the passage of particles smaller
than a pre-set size. The pre-set size can be selected as desired. A
very high volume flow of aerosol particles (e.g., up to 3313 Lpm)
can be drawn into the annular slot 401 formed in the cap 402 that
is designed to only allow the passage of particles smaller than 10
microns. The accepted particles continue on into a dichotomous
virtual impaction section 403 that returns all the aerosol
particles smaller than 1-micron back into the environment. The
remaining particles, (1-10 microns) are known as the product, flow.
The product flow continues into the next section.
[0063] As best illustrated by FIG. 5, a high volume flow of aerosol
particles is drawn into the annular slot 401 formed in the cap 402.
The annular slot 401 is designed to limit the upper or larger
particulate size range as they enter the collector. To efficiently
pass the smaller particulate, the cap 402 is a "passive" device in
that is has no moving parts and uses the fact that particulate with
a finite mass and moving in a flowstream (in this case air) will
not follow the streamlines exactly due to their inertia. If the
curvature of a streamline is sufficiently large and the mass of the
particulate is correspondingly high, the particle deviates far
enough from the streamline to impact with a surface. The particles
are drawn into the annular slot 401 and directed into the
transition section 409.
[0064] The APDS 300 has the capability to measure particle sizes in
the sampling environment via a built in particle counter with four
size ranges, and can store and display the results in real-time.
The system is entirely self-contained requiring only a 110 vac
power connection. The on-board computer has high-speed
communications capability allowing networks of these sampling
systems to be remotely operated.
[0065] The APDS 300 is useful for most environmental sampling. It
is particularly useful with biological material collection, but can
be used for collecting any airborne matter. The APDS 300 can be
used to sample air quality in public buildings such as convention
centers and sports arenas, for sampling in food processing
facilities, sampling animal pens (such as poultry houses), or for
use in monitoring orchards or agricultural areas for the presence
of pollens or pesticides. Because of it's relatively compact size
and weight it can be used to sample in confined spaces such as
found in aircraft or subway systems.
[0066] Referring now to FIG. 6, the virtual impactor section 403 is
shown in greater detail. In the virtual impactor section 403, the
separation efficiency is determined by the ratio of the major and
minor flows (or Bypass to Product) and the physical dimensions of
the nozzle and collection probe. The key is particulate larger than
the cut size become concentrated in the minor flow. The
concentration factor is the ratio of the total flow to the minor
flow. (If the minor flow is 25% of the total flow, then the
concentration factor is 4.) The aerosol passes through an
acceleration nozzle 601. The acceleration nozzle 601 has a diameter
D.sub.0. The aerosol is directed toward a collection probe 602. The
collection probe has a diameter D.sub.1. Between the acceleration
nozzle 601 and the collection probe 602, a major portion of the
flow 603 is diverted 90.degree. away. The minor or "product" flow
604 continues axially.
[0067] The flow forms streamlines 605. Small particles with low
inertia 606 follow the flow streamlines and are carried away
radially with the major flow 603. Large particles with greater
inertia 607 deviate from the flowlines but they continue moving
axially in their forward path down the collection probe 602 with
the minor or "product" flow 604. The separation efficiency is
determined by the ratio of the major and minor flows (or Bypass to
Product) and the physical dimensions of the nozzle D.sub.0 and
collection probe D.sub.1. The key is particulate larger than the
cut size become concentrated in the minor flow. The concentration
factor is the ratio of the total flow to the minor flow.
[0068] Referring now to FIG. 7, additional details of the sample
collection operation are shown. The particles, (1-10 microns) known
as the product flow are directed into a multi-stage, wetted-wall
cyclone collection section. In this stage of the sampling system
the product particles are trapped and concentrated into a liquid,
typically water, in a volume between 2 and 7 cc. An on-board
computer monitors and controls the flow of air through the system
using built in hot wire anemometers, as well as controlling the
liquid level in the cyclone. At a selected time the computer will
stop the flow of air and turn on a built-in peristaltic pump to
deliver the sample via an external liquid sample port.
[0069] The product flow particles enter a stainless steel funnel
section into the input of a multistage, wetted-wall cyclone
collector section 700. The system includes a cyclone collector 701,
peristaltic pump 707, an air pump 704, a vent 706, wash 705, 8
liter DD-H.sup.2O reservoir 702, and 1 liter bleach reservoir 703.
The reservoirs 702 and 703 are provided as external tanks outside
of the front panel interface 708. The multistage, wetted-wall
cyclone collector section 700 directs the particles of interest to
the sample preparation system 302.
[0070] The on-board computer monitors and controls the flow of air
through the system using built in hot wire anemometers that have
been mounted in the two exhaust ports of the sampler. The computer
and control software also act to control the liquid level in the
cyclone, and monitor all status indicators of the sampling system.
At a selected time the computer will stop the flow of air and turn
on a built-in peristaltic pump to deliver the collected liquid
sample via an external sample port. The system also has the
capability to measure particle sizes in the background environment
via a built in particle counter such as particle counter Biotest
APC-1000, with four size ranges, and can store and display the
results in real-time.
[0071] The system 300 is entirely self-contained requiring only a
110 vac power connection. The on-board computer has high-speed
communications capability allowing networking of multiple sampling
systems to be remotely operated. The computer has extra RS-232 or
RS-485 serial ports that can be used to control other
instrumentation. A keyboard, mouse, printer, displays, and other
peripherals can be "plugged" in at the rear of the system, or it
can be started "headless" (headless=Without a display, mouse,
etc.)
[0072] Referring now to FIGS. 8A, 8B, and 8C, the APDS Aerosol
Collection 301 and APDS In-Line Sample Preparation 302 sub systems
are shown in greater detail. The aerosol collection system 301 is
designated "High Collection Rate Aerosol Sampling System"
(HiCRASS). The HiCRASS comprises: Low Pass "Cap" 402; Transition
Section 409; Virtual Impactor 403; Funnel Section 410; Multistage,
Wetted-wall Cyclone Collector 700; Bypass Fan 412; and Control
Computer 714.
[0073] The HiCRASS system provides a very high volume flow of
aerosol particles (e.g., up to 3313 Lpm) that are drawn into the
annular slot 401 formed in the cap 402 that is designed to limit
the upper or larger particulate size range as they enter the
collector. The annular slot 401 allows the passage of particles
smaller than 10 microns. To efficiently pass the smaller
particulate, the cap 402 is a "passive" device in that is has no
moving parts and uses the fact that particulate with a finite mass
and moving in a flowstream (in this case air) will not follow the
streamlines exactly due to their inertia. The curvature of the
streamline is sufficiently large and the mass of the particulate is
correspondingly high that the particle deviates far enough from the
streamline to impact with a surface. The accepted particles
continue around the corner and onto the dichotomous virtual
impaction section 403 that returns substantially all the aerosol
particles smaller than 1-micron back into the environment.
[0074] The virtual impactor 403 works as the aerosol passes through
an accelerating nozzle 601 and is directed toward a collection
probe 602 where a major portion of the flow 603 is diverted
90.degree. away from it. The flow forms streamlines 605. Small
particles with low inertia 606 follow the flow streamlines and are
carried away radially with the major flow 603. Large particles with
greater inertia 607 deviate from the flowlines but they continue
moving axially in their forward path down the collection probe 602
with the minor or "product" flow 604. The separation efficiency is
determined by the ratio of the major and minor flows (or Bypass to
Product) and the physical dimensions of the nozzle D.sub.0 and
collection probe D.sub.1. Particulate larger than the cut size
become concentrated in the minor flow. The concentration factor is
the ratio of the total flow to the minor flow. (If the minor flow
is 25% of the total flow, then the concentration factor is 4).
[0075] The remaining particles (1-10 microns) now known as the
product 604, flow down a stainless steel funnel section into the
input of the multistage, wetted-wall cyclone collector section 700.
In this stage of the system 301 the product particles are trapped
and concentrated into a liquid, typically water, in a volume
between 2 and 7 cc. The wetted-wall cyclone collector section 700
is a system that causes the product flow particles 604 to be
collected by a liquid. The wetted-wall cyclone collector section
700 operates by forcing the air stream tangentially into a cylinder
causing the air stream to circulate around the inside of the
cylinder. Particles in the air stream having sufficient inertia
will collide with the interior wall where they are collected by the
liquid that circulates along the interior wall.
[0076] The on-board computer 714 monitors and controls the flow of
air through the system using built-in hot wire anemometers, as well
as controlling the liquid level in the cyclone 700. At a selected
time the computer 714 will stop the flow of air and turn on a
built-in peristaltic pump to deliver the sample via an external
sample port. The on-board computer 714 monitors and controls the
flow of air through the system using built in hot wire anemometers
that have been mounted in the two exhaust ports of the sampler. The
computer and control software also act to control the liquid level
in the cyclone, and monitor all status indicators of the sampling
system. At a selected time the computer will stop the flow of air
and turn on a built-in peristaltic pump to deliver the collected
liquid sample via an external sample port.
[0077] The system also has the capability to measure particle sizes
in the sampling environment via a built in particle counter such as
particle counter Biotest APC-1000, with four size ranges, and can
store and display the results in real-time. The system is entirely
self-contained requiring only a 110 vac power connection. The
on-board computer has high-speed communications capability allowing
networks of these sampling systems to be remotely operated.
[0078] Referring now to FIG. 9, another embodiment of the
collection section of the present invention is illustrated. This
collection section system is designated generally by the reference
numeral 900. The system 900 samples the air 901 and collects sample
particles of a predetermined particle size range from the air. The
system 900 is particularly useful with the latest generation of
Biological Warfare agent detection systems. An air sampling system
is a critical component in integrated biological warfare detection
system. The system 900 also has use in medical facilities and
research and development facilities.
[0079] A low pass section 902 has an opening of a preselected size
for gathering the air 901 but excluding particles larger than the
sample particles. In one embodiment, the opening of a preselected
size is an annular slot that only allows the passage of particles
smaller than 10 microns. The low pass section 902 produces a total
air flow 903 that contains the sample particles of a predetermined
particle size range. The low pass section 902 allows a very high
volume flow of air to be drawn through the preselected size
opening. In one embodiment, the very high volume flow of air is
3313 Lpm or less.
[0080] An impactor section 904 is connected to the low pass section
902 and receives the total air flow 903. The impactor section 904
separating the total air flow 903 into a bypass air flow 905 that
does not contain the sample particles and a product air flow 906
that does contain the sample particles. An accelerating nozzle and
a collection probe in the impactor section 904 diverts the bypass
air flow 90.degree. from the product air flow thereby separating
the bypass air flow and the product air flow. In one embodiment,
the bypass air flow and the product air flow separation is
determined by the ratio of the bypass air flow and the product air
flow. In one embodiment, the bypass air flow and the product air
flow separation is determined by the physical dimensions of the
accelerating nozzle and the collection probe. In one embodiment,
the bypass air flow and the product air flow separation is
determined by the ratio of the bypass air flow and the product air
flow and the physical dimensions of the accelerating nozzle and the
collection probe.
[0081] A wetted-wall cyclone collector section 907 is connected to
the impactor section 904. The wetted-wall cyclone collector section
907 receives the product air flow 906 and traps the sample
particles in a liquid. The sample particles of a predetermined
particle size range are concentrated in the liquid. In one
embodiment, the wetted-wall cyclone collector section 907 traps and
concentrates the sample particles into a liquid in a volume between
2 and 7 cc. In one embodiment, the liquid is water.
[0082] The system 900 is useful for most environmental sampling. It
is particularly useful with biological material collection, but can
be used for collecting any airborne matter. The system 900 can be
used to sample air quality in public buildings such as convention
centers and sports arenas, for sampling in food processing
facilities, sampling animal pens (such as poultry houses), or for
use in monitoring orchards or agricultural areas for the presence
of pollens or pesticides. Because of it's relatively compact size
and weight it can be used to sample in confined spaces such as
found in aircraft or subway systems.
[0083] APDS In-Line Sample Preparation--302
[0084] As best illustrated in FIG. 3, the in-line sample
preparation module 302 moves the sample from the aerosol collection
module 301 to appropriate modules within the APDS 300 and provides
sample preparation. In one mode, the sample preparation module 302
prepares the sample (mixing, filtering, incubation, etc.) and
delivers the sample reaction volume to the liquid-array based
multiplex immunoassay detection system 303. In another mode, the
sample preparation module 302 prepares the sample (mixing,
filtering, incubation, etc.) and delivers the sample reaction
volume to the in-line nucleic acid detection system 304.
[0085] The prior art sample preparation instrumentation uses
robotic manipulation of micropipettes coupled to disposable filter
wells. Robotics are inherently complex and difficult to scale. The
sample preparation module 302 uses Zone fluidics. 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. 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.
[0086] 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. 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.
[0087] The sample preparation module 302 uses a powerful, highly
flexible technique called sequential injection analysis (SIA).
Automation is achieved through the manipulation of small solution
zones under conditions of controlled dispersion in narrow bore
tubing. Zone fluidics makes use of a mufti-position selection valve
and a high precision, bi-directional pump to construct a stack of
well-defined sample and reagent zones in a holding coil of narrow
bore tubing. By appropriate manipulation of this zone stack, a wide
range of sample handling unit operations can be accommodated. The
pump is used to move the sample from one device to the next
achieving the required sample manipulation in the process. Once a
detectable species has been formed, the zone stack is transported
to the immunoassay detector 303 and to the nucleic acid detector
304.
[0088] Referring now to FIG. 10, a system for sample preparation
and detection is illustrated. The system is generally designated by
the reference numeral 302. The In-Line Sample Preparation 302 is
capable of performing, singly or in combination, Liquid-Array Based
Multiplex Immunoassay Detection 303 and/or In-Line Nucleic Acid
Amplification and Detection 304. The In-Line Sample Preparation
module 302 includes various components described below.
[0089] A means for injecting and or aspirating a sample 1001
provides injection and/or aspiration of the sample. In one
embodiment the injecting/aspirating means 1001 consists of a zone
fluidics system. In another embodiment the injecting/aspirating
means 1001 consists of an FIA system. The means 1001 for injecting
and or aspirating a sample can be, for example, a
injecting/aspirating device available under the trademark
milliGAT.TM. pump, Global FIA, Inc, Fox Island, Wash.
[0090] A means for adding a reagent to the sample 1002 is
operatively connected to the means 1001 for injecting and or
aspirating a sample. The means for adding reagent to the sample
1002 can be, for example, a unit for adding reagent to the sample
such as an injection or multi position selection valve, available
from VICI, Houston, Tex.
[0091] A means for mixing the sample and the reagent 1003 is
operatively connected to the means for adding reagent to the sample
1002. The mixing means 1003 mixes the sample with a reagent. The
means 1003 for mixing the sample and the reagent can be, for
example, a super serpentine reactor, available from Global FIA,
Inc, Fox Island, Wash.
[0092] A means for transporting the sample and the reagent 1004 is
operatively connected to the means for mixing the sample and the
reagent 1003. The means for transporting the sample and the reagent
1004 consists of a fluidics system. The means for transporting the
sample and the reagent 1004 can be, for example, FEP tubing
available from Cole-Parmer, Vernon Hills, Ill.
[0093] The Liquid-Array Based Multiplex Immunoassay Detection
module 303 measures a multiple pathogen targets in the sample. The
Liquid-Array Based Multiplex Immunoassay Detection module 303 will
be described in detail subsequently.
[0094] The In-Line Nucleic Acid Amplification and Detection module
304 provides a second detection system that is based on nucleic
acid amplification and detection. The In-Line Nucleic Acid
Amplification and Detection module 304 can be, for example, a
detection system described in publications and products produced by
Cepheid and Baltimore-based Environmental Technologies Group, Inc.
(ETG), a part of London-based Smiths Aerospace. The In-Line Nucleic
Acid Amplification and Detection module 304 will be described in
detail subsequently.
[0095] A means 1005 for transporting the amplified sample from the
Liquid-Array Based Multiplex Immunoassay Detection module 303 and
the In-Line Nucleic Acid Amplification and Detection module 304.
The means 1005 for transporting the amplified sample from the PCR
reactor can be, for example, FEP tubing available from Cole-Parmer,
Vernon Hills, Ill.
[0096] Conduits are included within the sample preparation module
302. Decontamination and conditioning the conduits is accomplished
by flushing the conduits with a suitable fluid. For example, the
decontamination and conditioning of all exposed conduits can be
performed by using a decontaminant, such as bleach, which is pumped
through the exposed conduits and then washed from the system with a
suitable wash solution.
[0097] The integrated remote control and feedback module 305 is
inherently autonomous, meaning control and/or monitoring functions
are ideally performed remotely. This networking of sensors can
occur in multiple different ways, from wireless solutions using RF,
to conventional hard-wired internet connections. Integrated remote
control and feedback module 305 is setup as a network of multiple
units to protect large areas, the higher sensitivity lowers the
number of required units. This reduces reagent and other associated
costs making deployment more feasible for a larger number of public
events. The integrated remote control and feedback module 305 is
statistically analyzed with a 1,000-sample aerosol sample library.
This library has been prescreened for the same pathogenic agents
used in the multiplex signatures. Therefore, any detection events
will serve as a final screen for incompatible primer pairing.
[0098] Detection--APDS Liquid-Array Based Multiplex Immunoassay
Detection--303
[0099] In operation of the APDS system 300, the aerosol collector
system samples the air, particles of a given size distribution are
trapped in a liquid, and a sample of interest is prepared. The next
step is detection of any pathogens in the sample particles. The
Liquid-Array Based Multiplex Immunoassay Detection system 303
measures a multiple pathogen targets in the sample. The
Liquid-Array Based Multiplex Immunoassay Detection system 303 has
the ability to use a detection modality that measures multiple
pathogen targets in the same sample. This prevents loss of
sensitivity due to sample dilution and severely reduces the
recurring assay cost for the instrument. "Liquid arrays" allow
optical or physical (i.e., shape, magnetism, etc.) encoding of
particles that then form the template for performing assays. In one
embodiment of the invention the Liquid-Array Based Multiplex
Immunoassay Detection system 303 uses Luminex technology. In other
embodiments, nanobarcodes--rod shaped structures on the nanometer
scale that can be optically barcoded with metal strips and then
measured via reflectance; quantum dots--encapsulation of nanometer
scale particles that emit specific light over a broad spectral
range; and upconverting phosphers are used. The liquid array
detection can occur as a preliminary screen, since it has the
capability to detect all types of pathogens (viruses, bacteria,
proteins, and spores) and is relatively low cost.
[0100] The Liquid-Array Based Multiplex Immunoassay Detection
system 303 uses a "liquid arrays," a highly multiplexed assay that
competes (in bead format) with "computer chip" platforms. In the
APDS system 300, the Liquid-Array Based Multiplex Immunoassay
Detection system 303 uses Luminex technology from Luminex
Corporation, Austin, Tex. The detection principle is built around
the use of optically encoded microbeads that can be used as assay
templates. Small diameter polystyrene beads are coded with 1000s of
antibodies. The sample is first exposed to the beads and the
bioagent, if present, is bound to the bead. A second, fluorescently
labeled antibody is then added to the sample resulting in a highly
fluorescent target for flow analysis. Since the assay is performed
on a microbead matrix, it is possible to measure all types of
pathogens, including viruses and toxins. Each microbead is colored
with a unique combination of red and orange emitting dyes. The
number of agents that can be detected from a single sample is
limited only by the number of colored bead sets. The system
includes the following components: microbead specific reagents,
incubation/mixing chambers, a microbead capture array, and an
optical measurement and decoding system.
[0101] The Liquid-Array Based Multiplex Immunoassay Detection
system 303 has sufficient precision to make a 10.times.10 array of
beads, making 100-plex bioagent detection viable. This can measure
a wide range of bioagents at sensitivities and selectivities
comparable to non-automated conventional immunoassay techniques
(such as enzyme-linked immunosorbent assays) that take 4-6 times as
long. Additional bead types are used as internal positive and
negative controls to monitor each step in the sample preparation
process. This provides quality control.
[0102] Confirmation--APDS In-Line Nucleic Acid Amplification and
Detection--304
[0103] In operation of the APDS system 300, the aerosol collector
system has sampled the air, particles of a given size distribution
have been trapped in a liquid, a sample of interest has been
prepared, and the detection system has detected a pathogen in the
sample. The next step is confirmation of the pathogen that has been
detected in the sample. The in-line nucleic acid amplification and
detection system 304, confirms the pathogen that has been detected
in the sample. PCR amplification devices are described in
publications such as 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 and many are commercially available such as ABI
PRISM.RTM. 7700 Sequence Detection System by Applied Biosystems;
iCycler iQ Real-Time PCR Detection System by Bio-Rad; and Smart
Cycler.RTM. System by Cepheid.
[0104] Referring now to FIGS. 11, 12, and 13, the liquid-array
based multiplex immunoassay detection system 303 is illustrated in
greater detail. FIG. 11 shows a 100-plex Luminex bead set 1100
generated by intercalating varying ratios of red and orange dyes
into polystyrene latex microspheres 1101. Each optically encoded
bead 1101 has a unique spectral address. The beads 1101 are shown
arranged so that they increase in red intensity in the vertical
axis and increase in orange intensity in the horizontal axis
providing the unique spectral address.
[0105] FIG. 12 shows the beads 1101 coated with capture antibodies
specific for target antigens. Examples of capture antibodies
include, anthrax 1102, plague 1103, small pox 1104, and botox 1105.
After incubating with the antigens, secondary or detector
antibodies are added, followed by addition of the fluorescent
reporter, phycoerythrin to complete the "antigen sandwich."
[0106] FIG. 13 shows the beads 1101 being analyzed in a flow
cytometer 1106. The beads 11-1 are interrogated one at a time. A
red laser 1107 (red) classifies the bead, identifying the bead
type. A green laser 1107 (green) quantifies the assay on the bead
surface, only those beads with a complete sandwich will fluoresce
in the green, and the signal is a function of antigen
concentration.
[0107] Referring again to FIG. 11, a set of 100 polystyrene
microbeads 1101 is shown. The beads are imbedded with precise
ratios of red and orange fluorescent dyes yielding an array of one
hundred beads, each with a unique spectral address. Each bead 1101
is coated with capture antibodies specific for a given antigen as
illustrated in FIG. 12. After incubating with the antigens,
secondary or detector antibodies are added, followed by addition of
the fluorescent reporter, phycoerythrin to complete the "antigen
sandwich."
[0108] After antigen capture, secondary antibodies sandwich the
bound antigen and are indirectly labeled by the fluorescent
reporter phycoerythrin (PE). Referring again to FIG. 13, each
optically encoded and fluorescently-labeled microbead is
individually interrogated by a Luminex flow cytometer 1106. A red
laser 1107 (red) excites the dye molecules imbedded inside the bead
and classifies the bead to its unique bead set, and a green laser
1107 (green) quantifies the assay at the bead surface. The flow
cytometer is capable of reading thousands of beads each second;
analysis can be completed in a little as 15 seconds.
[0109] Microbeads have several advantages over other solid-phase
supports such as planar waveguides or microtiter wells. First, the
5.5 (.+-.0.1) .mu.m spheres provide a large surface area that can
accommodate up to 100,000 capture antibodies per bead. The high
density of capture antibodies ensures maximum antigen binding,
thereby enhancing assay sensitivity. Second, because beads are
freely suspended in solution, the entire surface area is exposed,
increasing the probability of collisions with antigen in the proper
orientation for binding, facilitating rapid reactions. Agitating or
heating the reaction volume further improves reaction kinetics.
Also, the beads are effectively filtered on a filter-bottomed
plate. Filtration allows unbound antigen and other excess reagents
to be washed away, minimizing both non-specific binding and
undesired increases in background fluorescence.
[0110] The liquid-array based multiplex immunoassay detection
system 303 illustrated in FIGS. 11, 12, and 13 measures multiple
pathogen targets in the sample. Up to 100 different pathogens can
be detected in a single assay. Different antibodies on each bead
enables highly multiplex detection. Luminex bead-based assays that
are truly multiplexed; that is, assays designed for the
simultaneous detection of multiple threat agents using a single
sample. An example of a liquid-array based multiplex immunoassay
detection system is shown in U.S. patent application Ser. No.
2003/0003441 by Billy W. Colston, Matthew Everett, Fred P.
Milanovich, Steve B Brown, Kodumudi Venkateswaran, and Jonathan N.
Simon, published Jan. 2, 2003. The disclosure of U.S. patent
application Ser. No. 2003/0003441 is incorporated herein by
reference.
[0111] Referring now to FIG. 14, another embodiment of a system
constructed in accordance with the present invention is
illustrated. The system is designated generally by the reference
numeral 1400. The system 1400 comprises the following: Sample
Collection 1401, Sample Preparation 1402, Multiplex Amplification
PCR 1403, and Multiplex, Liquid Array Based Detection of PCR
Amplicon 1404.
[0112] The first stage of the system 1400 is "sample collection
1401" that provides collection of particles that could contain
targeted bioagents. The sample collection 1401 gathers air, water,
soil, or other substance being monitored. The sample collection
1401 separates selected potential bioagent particles from the air,
water, soil, or other substance.
[0113] The "sample preparation 1402" moves the sample from the
sample collection to appropriate modules within the system 1400 and
provides sample preparation. In one mode, the sample preparation
1402 prepares the sample (Lysis, Concentration, Purification,
Mixing, etc.) and delivers the sample to "Multiplex Amplification
PCR 1403." One mode provides "Multiplex, Liquid Array Based
Detection of PCR Amplicon 1404." An example of a flow cytometric
detection method for DNA samples is shown in U.S. patent
application Ser. No. 2002/0155482 by Shanavaz Nasarabadi, Richard
G. Langlois, and Kodumudi Venkateswaran published Oct. 24, 2002.
The disclosure of U.S. patent application Ser. No. 2002/0155482 is
incorporated herein by reference.
[0114] Referring now to FIG. 15, one specific embodiment of the
in-line nucleic acid amplification and detection system 1500 is
illustrated. The system 1500 is capable of performing, singly or in
combination, nucleic acid amplification, and nucleic acid detection
functions. The nucleic acid assay system 1500 includes a number of
components including means for injecting/aspirating a sample, 1501,
means for adding PCR reagent 1502, means for mixing sample and
reagent 1503, means for transport to PCR reactor 1504, means for
performing PCR amplification 1505, means for transport of amplified
sample from PCR reactor 1506, means for detection of PCR amplicon
1507, and means for decontamination and conditioning of all exposed
conduits 1508.
[0115] The means 1501 for injecting and or aspirating a sample
provides injection and/or aspiration of the sample. In one
embodiment the injecting/aspirating means 1501 consists of a zone
fluidics system. In another embodiment the injecting/aspirating
means 1501 consists of an FIA system. The means 1501 for injecting
and or aspirating a sample can be, for example, a
injecting/aspirating device available under the trademark
milliGAT.TM. pump, Global FIA, Inc, Fox Island, Wash.
[0116] The means 1502 for adding PCR reagent to the sample is
operatively connected to the means 1501 for injecting and or
aspirating a sample. The means 1502 for adding PCR reagent to the
sample can be, for example, a unit for adding PCR reagent to the
sample such as an injection or multi position selection valve,
available from VICI, Houston, Tex.
[0117] The means 1503 for mixing the sample and the reagent is
operatively connected to the means 1502 for adding PCR reagent to
the sample. The mixing means 1503 mixes the sample with a PCR
reagent. In one embodiment the PCR reagent includes primers. In
another embodiment the PCR reagent includes oligos. The means 1503
for mixing the sample and the reagent can be, for example, a super
serpentine reactor, available from Global FIA, Inc, Fox Island,
Wash.
[0118] The means 1504 for transporting the sample and the reagent
to a PCR reactor is operatively connected to the means 1503 for
mixing the sample and the reagent. The means 1504 for transporting
the sample and the reagent to a PCR reactor consists of a fluidics
system. The means 1504 for transporting the sample and the reagent
to a PCR reactor can be, for example, FEP tubing available from
Cole-Parmer, Vernon Hills, Ill.
[0119] The means 1505 for performing PCR amplification is
operatively connected to the means 1504 for transporting the sample
and the reagent to a PCR reactor. This results in an amplified
sample. In one embodiment the PCR amplification means 1505 includes
an embedded thermocouple calibration conduit. PCR amplification
devices are described in publications such as 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 and many are
commercially available such as ABI PRISM.RTM. 7700 Sequence
Detection System by Applied Biosystems; iCycler iQ Real-Time PCR
Detection System by Bio-Rad; and Smart Cycler.RTM. System by
Cepheid.
[0120] The means 1506 for transporting the amplified sample from
the PCR reactor is operatively connected to the means 1205 for
performing PCR amplification. The means 1506 for transporting the
amplified sample from the PCR reactor can be, for example, FEP
tubing available from Cole-Parmer, Vernon Hills, Ill.
[0121] The means 1507 for detection of PCR amplicon is operatively
connected to the means 1506 for transporting the amplified sample
from the PCR reactor. The means 1507 for detection of PCR amplicon
can be, for example, a detection system described in publications
and products produced by Cepheid and Baltimore-based Environmental
Technologies Group, Inc. (ETG), a part of London-based Smiths
Aerospace.
[0122] Conduits are included within the means 1501 for injecting
and or aspirating a sample, means 1502 for adding PCR reagent to
the sample, means 1503 for mixing the sample and the reagent, means
1504 for transporting the sample and the reagent to a PCR reactor,
means 1505 for performing PCR amplification, means 1506 for
transporting the amplified sample from the PCR reactor, and means
1507 for detection of PCR amplicon. A means 1508 for
decontamination and conditioning the conduits is directly connected
to the means 1507 for detection of PCR amplicon. The means 1508 for
decontamination and conditioning the conduits is operatively
connected to the means 1501 for injecting and or aspirating a
sample, means 1502 for adding PCR reagent to the sample, means 1503
for mixing the sample and the reagent, means 1504 for transporting
the sample and the reagent to a PCR reactor, means 1505 for
performing PCR amplification, means 1506 for transporting the
amplified sample from the PCR reactor, and means 1507 for detection
of PCR amplicon. The decontamination and conditioning of all
exposed conduits can be, for example, be performed by using a
decontaminant, such as bleach, which is pumped through the exposed
conduits and then washed from the system with a suitable wash
solution.
[0123] Referring now to FIG. 16, a block diagram illustrates
another embodiment of an autonomous pathogen detection system
constructed in accordance with the present invention. This
embodiment of an autonomous pathogen detection system is designated
generally by the reference numeral 1600. The autonomous pathogen
detection system 1600 provides water sample collection 1601, sample
preparation 1602, and detection 1603 and 1604.
[0124] In operation, a water sample collection unit 1601
continuously samples a water source. Water sampling systems are
known in the art. For example, a water sampling system is shown in
U.S. Pat. No. 6,306,350 issued Oct. 23, 2001 titled water sampling
method and apparatus with analyte integration. The disclosure of
U.S. Pat. No. 6,306,350 is incorporated herein by reference.
[0125] The in-line sample preparation unit 1602 concentrates the
sample in a swirling buffer solution. Particles of a given size
distribution are selected by varying the flow rate across a
separator unit. The in-line sample preparation system 1602 provides
all sample preparation steps (i.e., mix, wash, incubation, etc.),
and performing multiplex detection using a Luminex flow
cytometer.
[0126] In the "detection" sub-system 1603, a collected sample is
mixed with optically encoded microbeads. Each color of microbead
contains a capture assay that is specific for a given bioagent.
Fluorescent labels are added to identify the presence of each agent
on the bound bead. Each optically encoded and fluorescently labeled
microbead is individually read in a flow cytometer, and fluorescent
intensities are then correlated with bioagent concentrations.
[0127] In the "confirmation" sub-system 1604, PCR (nucleic acid)
amplification and detection confirms the presence of the bioagent.
An archived sample is mixed with the Taqman reagent, and then
introduced by a SIA system into a flow through polymerase chain
reaction (PCR) system. Specific nucleic acid signatures associated
with the targeted bioagent are amplified and detected using
fluorescence generated from nucleic acid replication from the
Taqman probes. In the "Integrated Remote Control and Feedback"
sub-system 1605, a central computer uses a simple serial based
Labview control system to control all instrument functions. A
software system provides data acquisition, real time data analysis,
and result reporting via a graphical user interface.
[0128] The first stage of the system 1600 is "water sample
collection 1601" that provides collection of particles from a
source of water that could contain bioagents. The water sample
collection system 1601 and in-line sample preparation 1602 provide
preconcentration and delivery of the particles of interest to a
wetted wall cyclone collector. The separator system captures
particles of interest.
[0129] In the wetted wall cyclone collector, the particles are
collected in a fluid, making downstream processing much easier. An
on board computer controls water flow rates and the size range of
particles collected. A particle counter provides reaptime feedback
on the size and quantity of particles collected.
[0130] Particles are drawn into the system that is designed to only
allow the collection of particles of a pre-set size. The pre-set
size can be selected as desired. The system is designed to only
collect particles that are desired. The accepted particles continue
on into a separator section that returns all the particles that are
not of the desired size back into the environment. The remaining
particles, are known as the product, flow. The product flow
continues into the detection sections.
[0131] The system 1600 has the capability to measure particle sizes
in the sampling environment via a built in particle counter with
four size ranges, and can store and display the results in
real-time. The system is entirely self-contained requiring only a
power connection. The on-board computer has high-speed
communications capability allowing networks of these sampling
systems to be remotely operated.
[0132] The 1600 is useful for many application of water sampling.
The system 1600 can be used to sample water quality in public
buildings, for sampling in food processing facilities, for use in
monitoring agricultural areas for the presence of pollens or
pesticides and other water sampling uses.
[0133] Referring now to FIG. 17, a block diagram illustrates
another embodiment of an autonomous pathogen detection system
constructed in accordance with the present invention. This
embodiment of an autonomous pathogen detection system is designated
generally by the reference numeral 1700. The autonomous pathogen
detection system 1700 provides soil sample collection 1701, sample
preparation 1702, and detection 1703 and 1704.
[0134] In operation, a soil sample collection unit 1701
continuously samples a soil source. Soil sampling systems are known
in the art. For example, a soil sampling system is shown in U.S.
Pat. No. 6,363,803 titled, vehicle mounted soil sampler invented by
Elmer Hubers, patented Apr. 2, 2002. The disclosure of U.S. Pat.
No. 6,363,803 is incorporated herein by reference.
[0135] The in-line sample preparation unit 1702 concentrates the
sample in a swirling buffer solution. Particles of a given size
distribution are selected by varying the flow rate across a
separator unit. The in-line sample preparation system 1702 provides
all sample preparation steps (i.e., mix, wash, incubation, etc.),
and performing multiplex detection using a Luminex flow
cytometer.
[0136] In the "detection" sub-system 1703, a collected sample is
mixed with optically encoded microbeads. Each color of microbead
contains a capture assay that is specific for a given bioagent.
Fluorescent labels are added to identify the presence of each agent
on the bound bead. Each optically encoded and fluorescently labeled
microbead is individually read in a flow cytometer, and fluorescent
intensities are then correlated with bioagent concentrations.
[0137] In the "confirmation" sub-system 1704, PCR (nucleic acid)
amplification and detection confirms the presence of the bioagent.
An archived sample is mixed with the Taqman reagent, and then
introduced by a SIA system into a flow through polymerase chain
reaction (PCR) system. Specific nucleic acid signatures associated
with the targeted bioagent are amplified and detected using
fluorescence generated from nucleic acid replication from the
Taqman probes. In the "Integrated Remote Control and Feedback"
sub-system 1705, a central computer uses a simple serial based
Labview control system to control all instrument functions. A
software system provides data acquisition, real time data analysis,
and result reporting via a graphical user interface.
[0138] The first stage of the system 1700 is "soil sample
collection 1701" that provides collection of particles from a
source of soil that could contain bioagents. The soil sample
collection system 1701 and in-line sample preparation 1702 provide
preconcentration and delivery of the particles of interest to a
wetted wall cyclone collector. The separator system captures
particles of interest.
[0139] In the wetted wall cyclone collector, the particles are
collected in a fluid, making downstream processing much easier. An
on board computer controls soil flow rates and the size range of
particles collected. A particle counter provides reaptime feedback
on the size and quantity of particles collected.
[0140] Particles are drawn into the system that is designed to only
allow the collection of particles of a pre-set size. The pre-set
size can be selected as desired. The system is designed to only
collect particles that are desired. The accepted particles continue
on into a separator section that returns all the particles that are
not of the desired size back into the environment. The remaining
particles, are known as the product, flow. The product flow
continues into the detection sections.
[0141] The system 1700 has the capability to measure particle sizes
in the sampling environment via a built in particle counter with
four size ranges, and can store and display the results in
real-time. The system is entirely self-contained requiring only a
power connection. The on-board computer has high-speed
communications capability allowing networks of these sampling
systems to be remotely operated. The 1700 is useful for many
application of soil sampling. The system 1700 can be used to sample
soil quality in monitoring agricultural areas for the presence of
pollens or pesticides and other soil sampling uses.
[0142] In operation of the pathogen detection system, the in-line
nucleic acid amplification and detection system provides nucleic
acid assay methods. The methods include a number of steps. One step
consists of automatically injecting and or aspirating a sample.
Another step consists of automatically adding PCR reagent to the
sample. Another step consists of automatically mixing the sample
and the reagent. Another step consists of automatically
transporting the sample and the reagent to a PCR reactor. The PCR
reactor consists of a fluidics system. Another step consists of
automatically performing PCR amplification resulting in an
amplified sample. Another step consists of automatically
transporting the amplified sample from the PCR reactor. Another
step consists of automatically detecting PCR amplicon. The method
is performed in a nucleic acid assay system and the nucleic acid
assay system is decontaminated and conditioned before a new sample
is analyzed.
[0143] The system includes both real time and post-PCR detection.
The system is ideal for monitoring type systems, such as those
currently being developed to detect terrorist releases of
aerosolized bioagents. On-site detection systems for infectious
diseases under development will need to incorporate sample
preparation and analysis functions. The system allows relatively
unskilled personnel, such as early responders, to perform real-time
field or point-of-care nucleic acid assays. In various other
embodiments of the autonomous pathogen detection system, the
confirmation of bioagent(s) in the sample is provided by a
multiplex immunoassay detector, a multiplex PCR detector, and a
real time PCR detector.
[0144] The present invention provides an Autonomous Pathogen
Detection System (APDS) for monitoring the environment to protect
the public from the release of hazardous biological agents. The
Autonomous Pathogen Detection System is a countermeasure to
bioterrorism, one of the most serious threats to the safety of
United States citizens, citizens of other countries, and the
military.
[0145] The APDS program was initiated to fill the requirement of a
distributed environmental monitoring system for civilian
applications. Multiplexed assays are used to reduce reagent costs,
making long term monitoring operations possible (e.g., U.S. Postal
Service mail screening). A unique, orthogonal detection approach
that combines antibody-based and nucleic acid-based assays reduces
false positives to a very low level. Antibody assays allow the
detector to respond to all types of bioagents, including those
without nucleic acids such as protein toxins. Nucleic acid assays
allow much more sensitive detection, reducing the number of sensors
needed to protect a given area. The fully autonomous aerosol
collection and sample preparation capabilities limit maintenance
requirements and makes integration into a central security or
monitoring network possible.
[0146] The Department of Transportation is actively seeking space,
providing monitoring biomonitoring systems for protection of
capabilities for Special Events, transportation hubs, with airports
residing at the facilities, transportation centers, top of this
list. The system is capable of meeting these needs.
[0147] There are other environmental or clinical pathogen detection
system needs. Mobile units could be transported to suspected "sick
buildings" to test for mold or fungal spores that might be causing
tenant illnesses. Units with reagents for animal diseases could be
placed in livestock transport centers or feedlots to rapidly detect
airborne pathogens and protect against disease outbreaks. Monitors
in hospitals could be used to test for airborne spread of
contagious materials among patients. The system could be used at
high profile events such as the Olympics for short-term, intensive
monitoring or more permanent installation in major public buildings
or transportation nodes. All of the individual units can be
networked to a single command center so that a small group of
technical experts can maintain and respond to alarms at any of the
units. The system is capable of meeting all of these needs.
[0148] The primary needs describe above are directed to protection
of civilians from terrorist attacks. The system also has uses in
protecting military personnel from biological warfare attacks. The
military continues to evaluate options to their current biowarfare
detection systems and the system meets many of the needs of the
military.
[0149] 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.
* * * * *