U.S. patent application number 10/441100 was filed with the patent office on 2004-04-01 for automatic point source biological agent detection system.
Invention is credited to DiFurio, Gabriel A., Schmidt, John C., Tilles, David J..
Application Number | 20040063197 10/441100 |
Document ID | / |
Family ID | 29584309 |
Filed Date | 2004-04-01 |
United States Patent
Application |
20040063197 |
Kind Code |
A1 |
Tilles, David J. ; et
al. |
April 1, 2004 |
Automatic point source biological agent detection system
Abstract
This invention is directed to a method and apparatus using
polymerase chain reaction (PCR) technology for automatically
collecting air samples and identifying biological agents in the air
sample. A fully automated system is provided that is capable of
detecting transient events such as bacillus anthracis in a piece of
mail being processed on high-speed mail processing equipment. The
system includes apparatus for implementing the following features:
particle collection and pre-separation using a collection hood and
dry cyclone passive filtration system; continuous particle
collection into a liquid sample; automated fluid transfer to a PCR
analysis cartridge at pre-scheduled times; automated cartridge
handling and transfer to PCR bio-agent identification apparatus for
detecting a bio-agent in a piece of mail; automatic retesting of
the liquid sample upon various error conditions; automatic
confirmation testing upon preliminary positive results; automated
fluid transfer to archive containers at the completion of analysis;
and, automated notification/reporting system to alert designated
personnel/organizations upon the occurance of selected events such
as the presence of bacillus antracis.
Inventors: |
Tilles, David J.;
(Woodstock, MD) ; DiFurio, Gabriel A.; (Baltimore,
MD) ; Schmidt, John C.; (Baltimore, MD) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
29584309 |
Appl. No.: |
10/441100 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60381351 |
May 20, 2002 |
|
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|
Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
G01N 2001/025 20130101;
G01N 35/0099 20130101; G01N 1/10 20130101; B01L 7/52 20130101; G01N
2001/022 20130101; G01N 1/4077 20130101; B01L 2400/0487 20130101;
G01N 1/24 20130101; B01L 2200/10 20130101; B01L 2300/0867 20130101;
G01N 2035/0436 20130101; G01N 1/2211 20130101; B01L 3/502 20130101;
G01N 2035/00881 20130101; G01N 1/2273 20130101; G01N 35/00871
20130101; G01N 2001/2217 20130101 |
Class at
Publication: |
435/287.2 |
International
Class: |
C12M 001/34 |
Claims
1. A fully automatic biological agent detection system, comprising:
biological agent identifier apparatus; collection apparatus for
collecting an aerosol sample of particles of an aerosolized
biological agent at at least one monitored location; aerosol
concentrator apparatus for producing a liquid sample of the aerosol
sample; an automated fluidics apparatus for storing and delivering
a portion of the liquid sample to a cartridge type receptacle; an
automated mechanical handling system for transporting the
receptacle from a staging area to a liquid fill point of the
fluidics apparatus and then to the biological agent identifier
apparatus; and control apparatus for providing overall automated
control of the system, for controlling the apparatus at said at
least one monitored location, and for reporting the test results
provided by the identifier apparatus to a predetermined
location.
2. The system according to claim 1 wherein the collection apparatus
operates continuously to collect samples of particles and wherein
the system for transporting operates periodically to transport a
receptacle from a staging area to a liquid fill point of the
fluidics apparatus and then to the biological agent identifier
apparatus.
3. The system according to claim 2 wherein the fluidics apparatus
periodically delivers a portion of the liquid sample to said
receptacle prior to the receptacle being transported to the
biological agent identifier apparatus.
4. The system according to claim 1 wherein the collection apparatus
includes a shroud/hood positioned at a collection point of the
monitored location.
5. The system according to claim 4 wherein the shroud/hood is
located along a mail transport path of mail processing
equipment.
6. The system according to claim 5 wherein the shroud/hood is
located at a mail pinch point along the mail transport path and
including mail pinching apparatus located adjacent the
shroud/hood.
7. The system according to claim 1 wherein the concentrator
apparatus includes a dry cyclone pre-separator and a wet-cyclone
aerosol concentrator assembly.
8. The system according to claim 1 wherein the fluidics apparatus
is capable of temporarily holding a sample in a reservoir where it
can be accessed for one or more analyses, while the aerosol
concentrator apparatus is collecting a next sample.
9. The system according to claim 1 wherein the fluidics apparatus
additionally archives a remaining portion of the liquid sample.
10. The system according to claim 1 wherein the mechanical handling
system includes a gripper type end effector for picking up and
transporting the cartridge type receptacle.
11. The system according to claim 10 wherein the staging area
includes a storage rack for holding a plurality of cartridge type
receptacles and wherein the end effector is located above the
storage rack.
12. The system according to claim 11 wherein the storage rack is an
elongatd rack holding the cartridge type receptacles in a linear
arrangement and wherein the end effector is located on and
automatically moves in two orthogonal directions on an elongated
track above the storage rack for grabbing, transporting and
returning one receptacle at a time, to and from the fluidics
apparatus and the biological agent identifier apparatus.
13. The sytem according to claim 12 wherein the fluidics apparatus
and the identifier apparatus are aligned with the storage rack so
that only two axes of motion are required of the end effector.
14. The system according to claim 13 wherein the end effector is
controlled by a machine controller forming part of said control
apparatus.
15. The system according to claim 14 wherein the machine controller
also controls the automatic operation of the fluidics apparatus and
an insert/extract cartridge mechanism forming part of the
biological agent identifier apparatus.
16. The system according to claim 1 wherein the biological agent
identifier apparatus comprises a polymerase chain reaction (PCR)
bioagent identifier.
17. The system according to claim 16 wherein the identifier
apparatus includes at least one screening cartridge containing an
assay for a single gene sequence of a target biological agent and
an internal control.
18. The system according to claim 16 wherein the identifier
apparatus comprises a single bay unit including automatic cartridge
exchange.
19. The system according to claim 1 wherein the control apparatus
includes a local machine control computer connected to and
controlling the operation of the biological agent identifier
apparatus, the aerosol concentrator apparatus, the fluidics
apparatus, and the mechanical handling system.
20. The system according to claim 19 wherein the control apparatus
additionally includes a remote site command and control computer
connected to and controlling the machine control computer as well
as communicating the test results to said predetermined
location.
21. The system according to claim 20 wherein the site command and
control computer is connected to the machine control computer via a
hardwire link.
22. The system according to claim 20 wherein the site command and
control computer is connected to the machine control computer via
an RF link.
23. The system according to claim 18 wherein the site command and
control computer include a primary and a backup control
computer.
24. The system according to claim 1 and additionally including a
cabinet for housing the aerosol concentrator apparatus, the
fluidics apparatus, the mechanical handling system, and the
biological agent identifier apparatus.
25. A method of detecting a biological agent in items to be
delivered and being transported along a transport path, comprising
the steps of: collecting an aerosol sample from said items at at
least one location of the transport path; producing a liquid sample
of the aerosol sample; delivering a portion of the liquid sample to
a cartridge type receptacle; mechanically transporting the
receptacle to biological agent identifier apparatus wherein said
identifier apparatus analyses the liquid sample for particles of a
predetermined biological agent; reporting the results of the
analyses provided by the identifier apparatus to a predetermined
location; and, providing overall automated control of the
method.
26. The method according to claim 25 wherein said items comprise
mail items.
27. The method according to claim 25 wherein the step of collecting
an aerosol sample includes the step of pinching said items at a
pinch point along the transport path.
28. The method according to claim 27 wherein the step of collecting
an aerosol sample includes locating an apparatus for pinching said
items and a shroud/hood for collecting aerosol samples pinched from
said items.
29. The method according to claim 28 wherein the step of
transporting comprises transporting a receptable containing the
liquid sample periodically to biological agent identifier
apparatus.
30. The method according to claim 29 wherein the step of
transporting includes using a mechanical handling system having a
gripper type end effector for picking up and transporting the
cartridge type receptacle under the control of a local machine
controller.
31. The method according to claim 30 wherein the step of
transporting includes periodically picking up and transporting a
cartridge type receptacle from a plurality of receptacles located
in a rack located beneath the end effector.
32. The method according to claim 28 whererin the step of
delivering a portion of the liquid sample includes the step of
temporarily storing the sample in a reservoir where it can be
accessed for one or more analyses.
33. The method according to claim 32 wherein the step of delivering
includes archiving a remaining portion of the liquid sample.
34. The method according to claim 25 wherein the biological agent
identifier apparatus comprisese a polymerase chain reactor (PCT)
bio-agent identifier.
35. The method according to claim 25 wherein the step of producing
a liquid sample includes using a dry cyclone pre-separator and a
web cyclone aerosol concentrator assembly.
36. The method of claim 25 wherein said step of providing control
of the method includes using local machine control computer
apparatus.
37. The method of claim 30 wherein said step of providing control
of the method also includes a remote site control computer
apparatus.
Description
CLAIM OF PRIORITY
[0001] This is a Non-Provisional application which claims priority
of the filing date of related Provisional Application Serial No.
60/381,351, filed on May 20, 2002, and which is incorporated herein
in its entirety by reference for any and all purposes.
BACKGROUND OF THE INVENTION
[0002] This invention is directed to biohazard detection systems
and more particularly to a biohazard detection system for detecting
biological agents, such as bacillus anthracis, in pieces of
mail.
DESCRIPTION OF RELATED ART
[0003] The current state of the art in biological agent detection
systems includes: (1) automated systems used, for example, by the
military that utilize a form of immunoassay technology; and (2)
manual systems including bio-identifier apparatus used in
laboratories by skilled laboratory technicians. The automated
immunoassay systems used by the military have not demonstrated
sufficient sensitivity or specificity to be acceptable for use in
civilian applications such as mail screening within the United
States Postal Service (USPS). Likewise, manual systems that require
skilled technicians to perform sample preparation and to interpret
test results are impractical in an industrial environment.
[0004] A typical automated bio-detection system in accordance with
the known prior art is comprised of the following subsystems: (a) a
trigger to detect the presence of a bio-agent and start the sample
collection process; (b) an aerosol collector for collecting samples
from the air; and, (c) an identifier to identify the specific
bio-agent.
[0005] In a manual detection system, a collected liquid sample is
manually taken from an aerosol collector, prepared, and introduced
manually into a bio-agent identifier. This process is time
consuming, hazardous, and can lead to erroneous results from
improper sample preparation.
[0006] In the USPS environment, various bio-detection systems have
been tested in connection with Mail Processing Equipment (MPE) but
have been found to be unreliable in distinguishing between letters
spiked with bacterial spores from uncontaminated letters or letters
containing hoax powders.
SUMMARY
[0007] Accordingly, it is the primary object of the subject
invention to detect an aerosolized biological agent in an aerosol
sample.
[0008] It is a further object of the subject invention to detect an
aerosolized biological agent originating from a piece of mail.
[0009] It is another object of the subject invention to provide a
biological agent detection system which achieves higher sensitivity
and lower false positives (false alarm) rates than current
technology.
[0010] The subject invention utilizes the polymerase chain reaction
(PCR) technology that is particularly adapted for USPS application.
The limit of detection for immunoassay based technology is in the
range of 10,000 to 100,000 spores per ml of sample. PCR has
demonstrated the ability to detect less than 200 spores per ml of
sample. This difference in sensitivity is critical, and may make
the difference between detecting and missing a lethal threat in the
USPS application. Since PCR detects the actual DNA sequence of an
agent, it is also, much less likely to cause false positives than
the systems based on immunoassay techniques.
[0011] This is achieved by an automatic biohazard detection system
(BDS) which combines automated fluidic transport apparatus with
aerosol collector apparatus and biological agent identifier
apparatus. The invention includes means for implementing the
following features: particle collection and pre-separation using a
collection hood and dry cyclone passive filtration system;
continuous particle collection into a liquid sample; automated
fluid transfer to a sample analysis cartridge at pre-scheduled
times; automated cartridge handling and transfer to polymerase
chain reaction (PCR) type bio-agent identifier apparatus for
detecting an actual DNA sequence so as to identify a bio-agent;
automatic retesting upon various error conditions; automatic
confirmation testing upon preliminary positive results; automated
fluid transfer to archive containers at the completion of analysis;
and automated notification/reporting system to alert designated
personnel/organizations upon the occurance of selected events. The
entire biohazard system is under control of a centralized command
and control computer.
[0012] The biological agent detection system in accordance with the
subject invention is not limited to, but is of particular
importance to the US Postal Service (USPS) due to the fact that it
would enhance the safety of its work force by quickly detecting the
presence of toxic biological agents in a mail processing facility.
The system would notify facility personnel so that appropriate
actions may be taken quickly to contain a threat from biological
agents, such as bacillus anthracis, in mail being processed at the
facility, thereby preventing dispersion of biological agents
between USPS facilities and to the general public.
[0013] The subject approach makes the system operation independent
of an optical trigger input. When desirable, however, an optical
trigger device may still be used, for example, to create a record
of particle concentration spikes that occur during the mail
processing window. This record will permit one to identify the
contaminated machine and the approximate time the contaminated
letter passed the machine after the identifier indicates that a
biological agent is present. In the future, if optical trigger
reliability improves, the subject system is compatible with the
integration of a trigger that operates in parallel with the
continuous collection process. In such an implementation, the
trigger would be used to initiate the transfer of the sample for
analysis, resulting in a more timely response to an incident.
[0014] Further scope of applicability of the present invention will
become apparent from the detailed description provided hereinafter.
It should be understood, however, that the detailed description and
specific example, while disclosing the preferred embodiment of the
invention, is provided by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The present invention will become more fully understood from
the detailed description provided hereinbelow and the accompanying
drawings which are given by way of illustration only, and
wherein:
[0016] FIGS. 1A and 1B are block diagrams illustrative of two
versions of a fully automatic bio-detection system control
architecture of a United States Postal Service (USPS) site in
accordance with the subject invention;
[0017] FIG. 2 is a block diagram illustrative of the apparatus
located at a USPS site in accordance with the subject
invention;
[0018] FIG. 3 is a system block diagram further illustrative of the
apparatus shown in FIG. 2;
[0019] FIGS. 4A-4D are illustrative of the location and mechanical
details of two types of aerosol sampling systems located at a mail
processing facility;
[0020] FIG. 5 is a front planar view of an illustrative embodiment
of the subject invention including an aerosol particle
concentrator, automatic fluidic sample preparation system, a
cartridge storage and handling system, and PCR identifier apparatus
in accordance with a preferred embodiment of the invention;
[0021] FIGS. 6A, 6B and 6C are perspective views respectively
illustrative of top and exploded views of a sample cartridge
utilized in connection with PCR identifier apparatus shown in FIG.
5;
[0022] FIG. 7 is a diagram illustrative of the operation performed
in the sample cartridge shown in FIGS. 6A-6C; and
[0023] FIG. 8 is a diagram illustrative of a flow chart of the
operation of the bio-detection system in accordance with the
subject invention.
DETAILED DESCRIPTION OF THE INVENTION
System Overview
[0024] Referring now to the various drawing figures, where like
reference numerals refer to like components throughout, a biohazard
detection system (BDS) 10 for a mail processing facility, such as,
but not limited to a United States Postal Service (USPS) site, can
monitor a single unit of mail processing equipment (MPE), or two or
more MPEs depending upon the configuration of the mail processing
system to be monitored. In the case of two or more bio-detection
systems, different system configurations are possible.
[0025] With respect to the block diagrams shown in FIGS. 1A and 1B,
the BDS 10 includes a plurality of monitoring units 12.sub.1 . . .
12n, (FIG. 1A) and 12.sub.1 . . . 12.sub.4 (FIG. 1B) whereas FIG. 2
is illustrative of a single monitor unit 12. In either case, one or
a plurality of the monitoring units 12 is under the control of a
central site command and control unit 14 which connects to an
external visibility and incident response network 16. In FIG. 1A, a
single site controller 15 is disclosed, while FIG. 1B discloses a
primary controller 151 as well as a backup controller 152.
[0026] As shown in FIG. 1A, each monitor unit 12.sub.1 . . .
12.sub.n is coupled to the site command and control unit 14 via a
hardwired network connection 18. In FIG. 1B, both a hardwired link
18 and an RF link 19 are utilized. Also, LAN (primary) and modem
(backup) communications implementation are provided. Each of the
monitor units 12.sub.1 . . . 12.sub.n includes three major
sub-systems under the control of a respective machine control
processor 20, namely: an aerosol collector/concentrator and
fluidics transfer sub-system 22, a cartridge handling sub-system
24, and a bio-identifier sub-system 26, to be described in greater
detail hereinafter.
[0027] A particle counter 28, as shown in FIG. 2, can also be added
when desired. The various sub-systems 22, 24 and 26 are located on
a common chassis including a cabinet shown by reference numeral
30.
[0028] Referring now collectively to FIGS. 3, and 4A-4D, each
monitor unit 12 of the subject BDS 10 includes a sampling hood 32
for sampling the air around one or more specific points, in this
instance a pinch point location 34 located in the mail transport
path 36 of high speed automated mail processing equipment (MPE).
FIG. 4A shows the transport path 36 of a facer/canceller system
used for canceling letters. Typical mail processing equipment such
as the facer/canceller transports mail items vertically by pinching
the letter between two belts 11 and 13 as shown in FIG. 4C. At the
pinch point location 34, the mail processing equipment switches
from a loosely held, non-singulated flow of mail pieces to a
singulated flow when a singulator 15 pinches an individual mail
piece and pulls it away from the non-singulated items. The location
of the sampling hood 32 at the pinch point location 34 is based
upon testing that demonstrates that particles contained in mail
pieces are expelled when the mail piece is pinched by the
singulator 15. The sampling hood 32 is configured to capture
virtually all of the particles expelled from the envelope at this
critical location. The sampling hood 32 includes a pair of side
channels 17.sub.1 and 17.sub.2 fixed on either side of the mail
path 31. The side channels 17.sub.1 and 17.sub.2 have cut-outs
19.sub.1 and 19.sub.2 to allow the mail transport belts 11 and 13
to pass through while still capturing the majority of the particles
expelled from the mail piece. A pair of gaskets 21.sub.1 and
21.sub.2 are located at the top of the side channels 17.sub.1 and
17.sub.2 to interface with a hinged hood 32. The hinged hood 32,
when in the lowered position, is the final element of a tunnel
consisting of the baseplate 23 of the mail processing equipment,
the two side channels 17.sub.1 and 17.sub.2 and the hinged hood 32.
The hinged hood has been shaped to guide the particles to the entry
point of the sampling hose 37 located at the downstream end of the
tunnel. The tunnel has been sized so that the sampling volume of
the aerosol concentrator (nominally 450 liters per minute) creates
sufficient face velocity of the air in the tunnel so that particles
in the inhalable threat region (up to 10 microns) will not settle
out inside the tunnel, but remain aerosolized. In addition, the
motion of the letters through the tunnel creates airflow through
the tunnel and mixes the air so that the particles do not settle
out within the tunnel and are available for sampling at the entry
point to the sampling hose 37 leading to the aerosol concentrator
22. The hood 32 is hinged to allow it to be lifted out of the way
to clear mail jams that sometimes occur at the singulator.
[0029] Alternate sampling systems have also been designed for other
pieces of mail processing equipment. In particular, a manifold
system 35 has been designed for a flat canceller as shown in FIG.
4D. This manifold system creates a downward airflow in the stacker
area 37 of the flats canceller. After the flats are cancelled, they
are stacked or placed back into an organized group so that they can
be placed into a container and transported to downstream
processing. As the flats sit in the stacker, a rotating arm 39
pushes against the flats to keep space available for the next flat
coming from the canceller. The rotating arm 39 repeatedly impacts
the flats sitting in the stacker, which has been shown to cause
particles in the flat mail piece to be expelled. These expelled
particles are then drawn down through the perforations in the
baseplate(s) 41, into the suction manifolds 43, and on through the
remaining components of the system. Similar sampling hood or
sampling manifold designs have been developed for other types of
mail processing equipment.
[0030] The first time that a letter, for example, is pinched at
pinch point location 34, air is pushed out of the envelope. If
there are particles inside the envelope, some will come out of the
envelope at that point. Sampling is performed within the hood 32
situated at location 34 by capturing the particles that are emitted
at the pinch point. The design of the hood and the sampling rate of
the air collector are matched so that the air inside the hood 32 is
sampled at a rate that will evacuate virtually all of the particles
present along this portion of the transport. This has two benefits,
namely: it reduces the dust that is created by the mail processing
operation, thereby reducing the cleaning maintenance required, and
it ensures that as many target particles as possible are captured
for analysis.
[0031] After the particles are captured, they are sent via a hose
37 through a dry cyclone 38, which is preferably located in the BDS
cabinet 30, as shown in FIG. 4B, that utilizes the particle
aerodynamic size to separate out larger particles, from those that
are in the inhalable size range, and therefore pose the highest
threat to human health. This cleans up the aerosol sample, and
prevents large dust and fibrous particles from clogging the
downstream equipment and interfering with the bio-detection
process. The large particles are captured in a container 40 and
disposed of. No filter media that can become clogged with dust is
utilized. The container 40 is also preferably located in the
cabinet 30.
[0032] The air from the pinch point 34 can, when desired, be
continuously monitored by an optional particle counter 28, as shown
in FIG. 3, which determines the number of particles per second in a
number of size ranges passing by the air sample point. The particle
counter 28 provides a historical record of particle count that may
assist one in identifying the contaminated mail sorting machine and
the approximate time a contaminated letter passed through the
machine in the event the monitor unit described below detects a
biological agent. If the particle counter 28 detects a spike in
particles with characteristics that match the target of interest,
such as bacillus anthracis, the system can use this event to
automatically trigger a sample analysis process to be described
hereinafter. Particle characteristics evaluated can include count,
size, shape, and fluorescence signature, among others. It is also
possible to use a mass spectrometer, not shown, as a trigger.
[0033] As noted, a BDS system in accordance with the subject
invention normally operates without a particle counter 28; however,
when utilized it can perform bio-agent analyses periodically based
upon the operational schedule formulated in the USPS facility.
[0034] Each of the monitor units 12.sub.1 . . . 12.sub.n, as noted
above, includes an aerosol collector/concentrator 22, an automated
sample cartridge handling system 24, and a bio-identifier 26 as
shown in FIG. 5.
[0035] Referring now to FIG. 5, an aerosol particle
collector/concentrator assembly 22, preferably a SpinCon.RTM.
system, constantly draws an air sample from the sampling hood 32
and impinges the sample into approximately 10 ml of liquid located
in a glass collector, not shown. At selected times derived from the
operational schedule formulated for the particular installation,
the solution is pumped out of the collector to a reservoir where it
is optionally mixed with a buffer liquid by one or more buffer
pumps 44. A fraction, nominally 2 ml, of the mixed sample is pumped
into one of the cartridges 46 at a fill station 48 after being
transported thereto by a two-axis end effector
(gripper/manipulator) 50 which forms part of the cartridge handling
system 24. Additional buffer and treatment solutions may also be
added, when desired, to the cartridge at the fill station 48.
[0036] The end effector 50 then automatically inserts the cartridge
46 into the biological identifier apparatus 26, preferably a
GeneXpert.TM. system instrument 47 that implements a polymerase
chain reaction (PCR) analysis capable of determining with a high
degree of reliability if any particles in the liquid sample
comprise a biological agent. The instrument 47 automatically
processes the sample and performs a PCR analysis to determine if
one or more biological agents are present. If the test result is
either positive for the agent(s) under test, or non-determinate,
indicating that certain internal controls included in the PCR
analysis did not perform correctly, an additional test is performed
using an additional fraction of the original sample and a new
cartridge. At the completion of the analysis, the remaining sample
is transferred from the reservoir into a waste bottle 52, or to
archive bottles 54 for later laboratory confirmatory analysis and
retention as evidence. The system can optionally individually
archive all samples or only those that generate a positive test
result.
[0037] The bio-identifier apparatus 24 is preferably controlled by
the remote central site command and control system 14 (FIG. 1) that
provides interface with the USPS network 16 but can be configured
for local control if desired.
[0038] The BDS continuously collects aerosol particles from
selected pinch point locations along the mail transport path 36 of
the MPE as shown in FIGS. 4A and 4B. Periodically, scheduled based
on the operating plan of the site, the liquid sample containing the
particles will be analyzed using an automated PCR test. This
initial analysis is termed a Preliminary, or Screening Test. If the
test is negative for agents of interest, no action is necessary,
and the facility operations will continue as usual.
[0039] If the result of the test is a "preliminary positive", the
system will automatically perform a confirmation (Reflex)-test,
optionally utilizing a criteria that is independent from the
Screening Test, such as a secondary gene sequence from the target
organism. Preliminary positive and confirmation test results are
reported to a Visibility/Incident Response network. The results can
be used to make the most appropriate decisions regarding personnel
evacuation and emergency response scenarios, and further analysis
of the archived sample using an outside laboratory. FIG. 8 is
illustrative of this sequence of events.
System Details
[0040] Site Control
[0041] Considering the subject invention in greater detail, the
site command and control system 14 (FIGS. 1A and 1B) provides
coordination and communication of all components in the overall
biohazard detection system (BDS). The site command and control
system 14 is designed to: (a) provide a single user interface to
the entire bio-detection system; (b) allow the user to quickly
determine the status of all components associated with the system;
and (c) accept input to change parameters which allow for the
configuration changes including options like frequency of testing
the collected sample, number of reports to print, passwords and
access levels, thresholds levels which display warnings for faults
and other system status. At its most basic level, the site command
and control system 14 provides an alarm when a positive reading has
been obtained from the bio-identifier instrument 47. The system 14
includes a control computer, not shown, that provides an interface
to the operators and supervisors about the status of the overall
system. This computer is furthermore networked to all sensor
devices (like particle counters) and to each monitor unit. 12. The
system 14 provides the higher level data collection of statistics
of each component that is necessary for reports and on screen
visibility. The system 14 also provides data about the test results
from the bio-identifier and relates particle counter data with each
PCR test occurrence and trends particle counter results over
specified periods of time.
[0042] Machine Control
[0043] Each monitor unit 12 contains a machine control processor 20
that sends and receives commands to and from the control computer
of site command and control system 14. The control processor 20
performs machine control functions which: (a) controls the fluid
interface between the collector/concentrator sub-system 22 and the
bio-identifier sub-system 26; and (b) responds to any faults or
alarms from the collector/concentrator 22 and the bio-identifier
subsystem 26.
[0044] Machine control functionality provided by the processor 20
has been separated from the site command and control computer 14
because the machine control processor 20 handles time critical
commands that affect the operation of the system components in the
monitor unit 12 and which includes the aerosol
collector/concentrator sub-system 22, the cartridge handling
sub-system 24, and the bio-identifier sub-system 26.
[0045] Aerosol Collector/Concentrator
[0046] Several different types of aerosol collector/concentrators
22 can be used with the subject system, however, the preferred
embodiment of this equipment comprises a proprietary SpinCon.RTM.
system developed by Midwest Research Instititute (MRI). The
SpinCon.RTM. apparatus 22 is an efficient device proven to be well
suited for a broad range of advanced air sampling requirements,
including the collection of bio-aerosols, particulate matter, and
soluble vapors. The primary sample collection component of the
SpinCon.RTM. system 22 consists of a vertical glass tube, not
shown, open on the top end, with a nearly tangential, vertical slit
cut into the side and is called the contactor. Fluid is placed in
the contactor and air is drawn through the slit and out through the
open top end of the contactor. The slit acts like a venturi/air
blast atomizer; as the air passes through the slit, it speeds up
and then impacts the spinning fluid in the contactor forming a wet
cyclone. The collection fluid then atomizes into many small
droplets, greatly increasing the surface area in contact with the
air. These droplets then begin to follow the air path. The slit is
only nearly tangential so the air path is across a chord of the
contactor's circular cross-section. At this time, particles in the
air are picked up by the fluid. As the air and droplets reach the
other side of the contactor, the droplets impinge on the wall and
the fluid flow is reformed. The same fluid is re-atomized over and
over, thus causing the concentration of particles in the fluid to
increase linearly with time. The spinning fluid in the contactor
only covers 30 to 40 percent of the slit, which is why only 30 to
40 percent of the air is sampled that is pulled into the unit.
[0047] The SpinCon.RTM. system 22 is very effective in collecting
biologicals (sizes 1-10 microns) as well as many types of smaller
particles and even chemicals (agglomerated sizes <1 micron.)
This is due to the atomized state of the fluid at the point of
collection; the massive surface area collects the larger particles,
while Brownian motion, which governs the motion of small particles,
enables the smaller particles to be picked up in the fluid.
[0048] Cartridge Handling Automation:
[0049] The cartridge handling sub-system 24 as shown in FIG. 5
mechanically links the apparatus of the collector/concentrator
subsystem 22 with the PCR test instrument 47 of the bio-identifier
subsystem 26. In addition to the two-axis end effector or
manipulator assembly 50 which includes a gripper 52 for securely
holding a cartridge 46, the cartridge handling sub-system 24 also
includes a track 54 which is located over a cartridge storage rack
56 which holds a predetermined number of liquid sample cartridges
46 so as to provide up to 10 hours or unattended operation. The
processor 20 (FIG. 3) includes a controller that executes the
cartridge handling process and interfaces with the rest of the
subject BDS to coordinate the sample transfer and identification
processes.
[0050] The two-axis end effector/manipulator 50 shown in FIG. 5
comprises a mechanical assembly which is a simple, inexpensive
design that has a form-factor that favors the footprint of the
bio-detection apparatus 26 (long X short Y). It shares the same
cabinet 30 as the collector/concentrator 22 and bio-identifier 26,
resulting in a compact, integrated detection system.
[0051] Additional features of the Cartridge Handling System 24
include: (a) stepping motor operates with position feedback; (b) it
simultaneously presents liquid sample cartridges 46, one at a time,
to three hypodermic needles, two of which, 60 and 62, are shown in
FIG. 5, for sample and wash buffer fill, with no additional axes of
motion; (c) no direct operator interface; and (d) controller
interface to higher level control; and (e) insert/extract mechanism
64, to the bio-identifier instrument 47.
[0052] Bio Identifier:
[0053] As noted above, two technologies are commonly used in the
detection of biological warfare agents: namely, (1) immunoassay and
(2) polymerase chain reaction (PCR). Immunoassay technology is
based on the specific interaction of antibodies with pathogen. This
interaction is usually detected optically or electrochemically.
PCR, on the other hand, directly detects the DNA sequence of an
agent.
[0054] PCR technology has been selected for the subject invention
because of its superior sensitivity and specificity. The limit of
detection for immunoassay based technology is in the range of
10,000 to 100,000 spores per ml of sample. PCR has demonstrated the
ability to detect less than 200 spores per ml of sample. This
difference in sensitivity is critical, and can make the difference
between detecting and missing a lethal threat, for example, in a
USPS application. Since PCR detects the actual DNA sequence of an
agent, it is also much less likely to cause a false positive than
the systems based on immunoassay techniques. Also, sequences
associated with the actual virulence properties of the organism can
be targeted. This will also be critical for a USPS application,
since a false positive may result in a major financial loss if it
causes an unnecessary shutdown of a mail processing facility.
[0055] PCR techniques have become recognized as one of the most
reliable laboratory techniques, along with culture methods, to
validate immunoassay and other field screening techniques. In
recent years the development of real time PCR techniques have
allowed the reaction to be performed in 30 minutes or less. This
enables the use of PCR in field applications where rapid results
are required. However, all current PCR methods require sample
preparation to remove inhibitors (such as the humic acids from
soil) from the sample that may result in a false negative and add
reagents necessary to run PCR. This sample processing requires
significant laboratory operations that USPS personnel could not
reliably perform in the current mail processing facilities. For
this reason, most PCR systems, cannot be used in the USPS
application or similar industrial environments.
[0056] The subject invention uses a PCR bio-identifier system that
completely automates both sample processing and detection
processing and comprises a GeneXpert.TM. system developed by
Cepheid of Sunnyvale, Calif. This system consists of two
components, a disposable multi-chamber cartridge such as shown in
FIGS. 6A-6C by reference numeral 46 and a PCR analysis instrument
47. The aerosol collector 22 described previously automatically
loads a liquid sample into a GeneXpert.TM. cartridge 46 which is
then transported to the GeneXpert.TM. instrument 47 by the
manipulator assembly 50. The GeneXpert.TM. instrument 47 then
automatically performs the entire sample preparation, PCR
amplification, and results analysis with no additional intervention
by the user. The fluid sample and liquid reagents are automatically
transported from one chamber 60 (FIG. 6B) to another within the
disposable cartridge as shown in FIG. 7. Fluids are mixed,
molecules and organisms are separated, purification is
accomplished, filtering is performed, lysing is completed, all
automatically with no operator intervention. The GeneXpert.TM.
system automates all fluidic processing steps.
[0057] The key advantages of the GeneXpert.TM. bio-identifier
system 26 utilized in the subject invention are:
[0058] (a) on-board PCR reagents--The critical PCR chemicals (or
reagents) are "on-board" the GeneXpert.TM. cartridge 46, and are
installed at the factory. Thus, the operator does not need to
handle the sensitive reagents. Since they are pre-mixed and
lyophilized at the factory, there is no chance for mistakes in
mixing by an operator and thus there is no need to refrigerate the
cartridges;
[0059] (b) spore lysing--The GeneXpert.TM. instrument 47
incorporates an ultrasonic lysing region which actually cracks open
the spore, releasing the DNA from inside the organism, in about 15
seconds. This capability does not exist with any other known DNA
analysis system. Systems that do not lyse the organism cannot
guarantee that the DNA from the organism is actually available for
PCR detection. Such systems that do not lyse can readily report a
false negative, especially for spores such as bacillus
anthracis;
[0060] (c) inhibitor removal--Many types of common biological
samples, including common dirt, contain extraneous chemicals that
impede the PCR detection reaction. The presence of these inhibiting
chemicals can cause PCR reaction to fail, thereby resulting in a
false negative. The GeneXpert.TM. instrument 47 captures the
spores, then actually washes them with a PCR-compatible buffer
solution to remove any potential inhibiting chemicals prior to
performing the PCR reaction itself. Systems which do not remove
inhibitory chemicals can easily report a false negative;
[0061] (d) pathogen concentration--Pathogens can be present in raw
samples or can be released into the air at extremely low
concentrations, yet still remain infectious. In order to ensure
that such pathogens can be detected with the highest possible
sensitivity, the GeneXpert.TM. instrument 47 actually extracts and
concentrates the spores from a relatively large original sample
volume (up to several mL) into a small PCR reaction tube of the
cartridge 46. Other PCR instruments simply take a small portion of
the available liquid sample and perform PCR on this small portion.
As a result of the concentrating ability of the GeneXpert.TM.
apparatus 47, the system routinely achieves a sensitivity at least
10 times better than competitive products which do not concentrate
the sample;
[0062] (e) no environmental contamination or cross
contamination--Since all the fluidic activity for PCR detection
occurs automatically and is completely contained inside the
GeneXpert.TM. cartridge 46, it is impossible for the GeneXpert.TM.
instrument 47 to inadvertently contaminate the environment or the
instrument with PCR product. For example, if a specific sample
tests positive for bacillus anthracis, the resulting liquid is now
very concentrated with bacillus anthracis DNA. In a manual-based
system, small portions of this liquid could escape into the
environment as liquids are pipetted or moved from tube to tube. If
bacillus anthracis DNA from the PCR reaction escapes into the
environment, this could become a source of contaminating DNA which
could cause a false positive during subsequent tests. Since fluids
are always retained inside the GeneXpert.TM. cartridge 46, such
potential false positives are eliminated;
[0063] (f) robust reaction tubes--GeneXpert.TM. cartridges 46 and
integrated reaction tubes 60 as shown in FIG. 6B are all plastic.
In contrast, other products have glass reaction tubes. These glass
tubes easily break. When they do break, they not only present a
maintenance, service, and reliability issue, but they can also
contaminate the environment with bacillus anthracis DNA, again
providing a source for potential false positives during subsequent
tests; and,
[0064] (g) multi-target detection--When using PCR, the definitive
identification of bacillus anthracis, for example, requires the
detection of two different DNA segments. The GeneXpert.TM.
instrument 47 has a versatile multiplexing capability in that
multiple DNA targets can be detected simultaneously in the same PCR
reaction tube 60 of a cartridge. Multiplexing capability is a
critical feature for DNA analysis and pathogen detection. For
example, with the GeneXpert.TM. system, a single test or analysis
for up to four agents can be performed within a single disposable
cartridge 46. Alternatively, a completely confirmatory test for an
agent such as bacillus anthracis can be performed within a single
cartridge 46. This assay would include three probes for the three
different DNA segments and one probe for an internal control. With
the GeneXpert.TM. instrument 47, this can be done in a single test
cartridge 46. Finally, most robust PCR chemistries require an
internal "control" DNA sequence. This control sequence is amplified
and detected along with the "target" DNA (such as bacillus
anthracis) to assure that the PCR chemistry is performing
properly--basically a validation or quality check. The
GeneXpert.TM. instrument 47 has four independent optical detection
channels. Accordingly, these advanced, but necessary, multiplexing
chemistries can be utilized for: (1) multiple pathogen detection;
(2) confirmatory testing; and/or (3) test quality/validation
control.
[0065] In current PCR methods, separate positive and negative
controls must be run to assure reagent integrity or successful
removal of inhibitors during sample preparation. A new internal
control scheme that eliminates the need for these external controls
is achieved by a unique combination of an internal control and
probe integrity check called probe check. The internal control
consists of a piece of DNA whose sequence is different than the
target DNA and a corresponding probe that is included in the PCR
bead. The internal control is co-amplified along with the test
reaction and is used to assure that the reagent is functional and
that PCR inhibitors have been successfully removed during sample
preparation.
[0066] System Operation
[0067] In a USPS installation, the biological agent detection
system (BDS) in accordance with the subject invention is deployed
on mail processing equipment (MPE). The operation of the subject
bio-detection system is controlled by the machine control processor
20, and its operation is synchronized with the operation of the
monitored MPE so that it is only allowed to operate when the BDS
collector/concentrator is operational. The flow chart shown in FIG.
8 illustrates the operational sequence.
[0068] Prior to collecting samples, the BDS must be initialized and
prepared for data collection. The following describes the daily
setup tasks: (1) start-up of site command and control system; (2)
set collection parameters for the day. The collection parameters
include the setup for each run in sequential order for the tour.
The run setup will indicate the machine ID sample number, start
time, stop time, and the assay description. The assay description
is associated with a command sequence used by the GeneXpert.TM. to
perform the PCR analysis. The command sequences are stored locally
in the machine control processor 20 (FIG. 3). The supervisory
console 14 (FIGS. 1A and 1B) will have the capability to download a
new assay description and associated command sequence to the
machine control processor; and, (3) powers up the BDS cabinets. The
system will automatically perform a communications and systems
status check; rinse and prime the fluid lines; and indicate whether
fluid levels are low.
[0069] At the specified start time, the BDS initiates the air
collection process. This enables the collector/concentrator
sub-system 22 to start operation. An indicator, not shown, on the
cabinet 30 (FIG. 5) provides an indication that the system is
active.
[0070] Air is then sampled from one or more areas 34 of the MPE as
shown in FIGS. 4A and 4B. The sampling area(s) have been
empirically determined based on tests that located areas where high
volumes of particles come out of the mail pieces as they are
processed. The output of the air collection hood 32 is routed via
tube 37 which is a grounded anti-static tube to the dry cyclone
pre-separator 38 that is designed to eliminate particles that are
larger than the inhalation threat range of 1-10 microns.
[0071] From the dry-cyclone 38, the sampled aerosol is routed via
40 to the SpinCon.RTM. collector/concentrator apparatus 22 (as
shown in FIG. 5). As noted above, the apparatus 22 impinges the air
into a small volume of liquid. The aerosol collector operates at a
flow about 450 lpm. As air passes through the unit, cyclonic mixing
transfers a high portion of the target particles into the liquid.
The liquid medium remains in the collector/concentrator 22 to
continuously concentrate the target particles into the liquid. At
the start of the collection process, 10 ml of sterile water is
injected into the system. During the collection, the water level is
monitored, and evaporated water is replaced by injecting makeup
water to maintain to 10 ml sample volume.
[0072] At a planned "stop time" or in response to a trigger input,
the machine control processor 20 sends a signal to the
collector/concentrator 22 to transfer a sample out for analysis.
The aerosol collection process and facer/canceller operation are
paused while the sample is transferred into the collection
reservoir 43 (FIG. 5), and the collector/concentrator 22 is then
refilled to start the next collection window.
[0073] As the liquid sample is transferred into the reservoir 43,
it is mixed with a solution containing additives that minimize PCR
inhibition. The liquid sample is then allowed to sit in the
reservoir for a time, e.g., approximately two minutes, to allow
thorough mixing of the additive solution, and allow any large
particles to settle to the bottom of the reservoir bottle(s)
42.
[0074] While the liquid is setting, the gripper mechanism 52 of the
end effector 50 of the cartridge handling system 24 as shown in
FIG. 5 retrieves a PCR cartridge 46 from the storage rack 56, and
places it in position at the "liquid fill" station 48 in the BDS
cabinet as shown in FIG. 5. The three needles at the liquid fill
station 48, two of which are shown by reference numerals 60 and 62,
pierce a seal on the top of the cartridge 46, and allows the sample
and wash buffer solutions to be added to the appropriate cartridge
chambers.
[0075] The liquid transfers are performed utilizing peristaltic
pumps 44. Once the sample transfer is complete, the cartridge 46 is
placed into the GeneXpert.TM. instrument 47, and the sample
analysis process is started.
[0076] After the cartridge 46 is inserted into the GeneXpert.TM.
instrument 47, an automated sample preparation process begins. The
sample is concentrated, washed, sonicated, mixed with the PCR
reagents, and moved into a reaction tube 60 (FIG. 6B) for PCR
thermal-cycling as shown in FIG. 7. Each of these steps, along with
the parameters that control the PCR analysis itself, is elaborated
in an assay file that is specific to the test being performed.
[0077] Tests
[0078] After the sample preparation steps are complete, PCR thermal
cycling analysis begins. The primary PCR test is called a Screening
Test. This test targets one or more gene sequences for each of the
organisms of interest. In addition to the target organisms, the
Screening Test also includes an internal control signal that
provides a built-in positive control that the PCR reaction has
proceeded properly. As the PCR thermal cycles are performed, the
fluorescence signals in the cartridge reaction chamber are
monitored and analyzed on each thermal cycle using an algorithm
that analyzes the shape of the PCR growth curve, including features
such as its cycle threshold and endpoint to deteermine whether the
PCR result indicates the presence of the target organism.
[0079] (Screening Negative)--In normal conditions, the test results
of the Screening Test are negative (N). The test results are sent
to the site command and control system 14 (FIG. 1) where the
results are logged. The test cartridge 46 is removed from the
GeneXpert.TM. instrument 47 and placed back into its position on
the cartridge storage rack 56. The remaining liquid sample in the
reservoir bottle(s) 42 is transferred to an archive bottle 54 (or
optionally to a waste bottle 52 if the "archive all" parameter is
turned OFF). The SpinCon.RTM. reservoir 43 is then available for
the next sample.
[0080] (Screening Positive/Preliminary Positive)--If the PCR
bio-identifier instrument 47 detects a positive (Y) Screening Test
result, the results are sent to the site command and control system
14, where notifications are sent out according to a prescribed
notification and response scenario and a Reflex Test is next
performed as will be described hereinafter.
[0081] (Screening Process Error/Inhibition)--If the PCR
bio-identifier instrument 47 detects an invalid screening result,
the test results are also sent to the site command and control
system 14, where notifications are sent out again, according to a
prescribed notification and response scenario. The system has the
capability of utilizing an alternate assay for the repeat test
based on the nature of the error on the original screening test.
If, based on the background fluorescence, it appears as if there
was a bead rehydration or other processing problem, a portion of
the archived sample will be utilized to repeat the same assay in a
new cartridge 46. If the error appears to be an inhibited sample, a
portion of the archived sample will be utilized to perform a
slightly modified assay. This assay will: (1) perform additional
washes; (2) utilize a higher level of dilution; and (3) adjust the
positive detection thresholds based on the modified dilution.
[0082] (Reflex Test)--In response to a positive (Y) Screening Test
result, (a) the site command and control system 14 will send out
Preliminary Positive notifications to the designated contact list,
(b) the cartridge handling system 24 (FIG. 5) will retrieve the
cartridge to be used for the Reflex Test, and transport it to the
fill station where a fraction of the sample remaining in the
reservoir and buffer solutions are transferred into it, and
depending on the agents to be tested for, the Reflex Test may
simply consist of a repeat of the Screening Test, or it may be
performed on a special "reflex" cartridge 46' containing primers
for alternate genetic sequences, (c) the appropriate assay for the
reflex cartridge is selected, and (d) the reflex cartridge 46' will
then be automatically loaded into the GeneXpert.TM. instrument 47
and a Reflex analysis will be performed.
[0083] (Reflex Negative)--The system will transfer the remaining
liquid sample into an archive tube 54. For a negative (N) Reflex
Test result, no site alarms or emergency response action are
initiated, the GeneXpert.TM. test results are sent to the site
command and control system 14, where the results are logged and
test result notifications are sent out. The original screening
cartridge, the reflex cartridge, and the archive tube can
optionally be manually retrieved from the system and saved in
refrigerated storage for further analysis to determine the cause of
the preliminary positive.
[0084] (Reflex Process Error/Inhibition)--For a Reflex Process
Error/Inhibition result, no local alarms or emergency response
actions are initiated, the test results are sent to the site
command and control system 14, where the results are logged and
notifications are sent out according to a prescribed notification
and response scenario. Another reflex test can be performed, as
long as sufficient sample is available.
[0085] (Reflex Positive)--The system will transfer the remaining
liquid sample into an archive bottle 54. For a positive (Y) Reflex
Test result, the GeneXpert.TM. test results are sent to the site
command and control system 14, where the results are logged and
test result notifications are sent out. The site emergency response
plan is put into effect.
[0086] Thus what has been shown and described is a unique biohazard
detection system for detecting toxic biological agents,
particularly bacillus anthracis, in a facility which, for example,
handles and processes items, such as mail.
[0087] The detailed description provided above, however, merely
illustrates the principles of the invention. It will thus be
appreciated that those skilled in the art will be able to devise
various arrangements which, although not explicitly described or
shown herein, embody the principles of the invention and are thus
within its spirit and scope.
* * * * *