Rapid Bacterial Detection Rapid Bacterial Detection SystemSystem
Fully Automated 4 minute CFU/ml response Fully Automated 4 minute CFU/ml response systemsystem
SUBC Inc.
Rochester MN
MII Inc. ConfidentialMII Inc. Confidential
Principle and Sequence of Principle and Sequence of Rapid DetectionRapid Detection
1. Platelets and cellular debris removed from 1. Platelets and cellular debris removed from platelet concentrate sampleplatelet concentrate sample
2. Present Bacteria are isolated and immobilized 2. Present Bacteria are isolated and immobilized in isolation chamberin isolation chamber
3. Isolation chamber rinsed with buffer, leaving 3. Isolation chamber rinsed with buffer, leaving isolated bacteriaisolated bacteria
4. Lyse solution in combination with localized 4. Lyse solution in combination with localized heat provide rapid bacterial membrane lyseheat provide rapid bacterial membrane lyse
5. Bacterial ATP containing lysate is mixed with 5. Bacterial ATP containing lysate is mixed with bioluminescent firefly extractbioluminescent firefly extract
6. Generated light reaction is detected by photon 6. Generated light reaction is detected by photon counter. Burst of light is correlated to CFU/mlcounter. Burst of light is correlated to CFU/ml
Fluidic Analysis for Fluidic Analysis for Detection of BacteriaDetection of Bacteria
Sampling Sample Processing Detection
Carrier Stream
DilutionMixingEnrichmentChemistry
Waste
Pump
1 2 3
4
5
678
9
10 Selectionvalve
Carrier
Detector
ReactorHoldingcoil
Sample
Reagent
SIA
Sequential Injection Sequential Injection Analysis for Bacterial Analysis for Bacterial
DetectionDetection
CFURapid
Platelets
RinseLyse
+Heat
Platelet removalSystem
Bacteria Isolation Chamber
wasteReactor
Rapid Fluidic Engine Rapid Fluidic Engine SequenceSequence
3 Minute Result
BioluminescentReagent
Firefly Luciferace Firefly Luciferace ReactionReaction
ATP +D-luciferin + O2 AMP+pyrophosphate+oxyluciferin+CO2 + Light
TheThe quantum efficiency is very high resulting in quantum efficiency is very high resulting in almost one photon per ATP molecules consumed almost one photon per ATP molecules consumed in the reactionin the reaction
Intensity of the emitted light is proportional to Intensity of the emitted light is proportional to the ATP concentrationthe ATP concentration
If the luciferase level is low the intensity will be If the luciferase level is low the intensity will be essentially constantessentially constant
If the luciferase level is high the light will decay If the luciferase level is high the light will decay rapidly since ATP is consumed in the reaction (the rapidly since ATP is consumed in the reaction (the initial peak light is proportional to the decay rate)initial peak light is proportional to the decay rate)
If decay rate t If decay rate t 1/2 1/2 = 139 minutes sensitivity range = 139 minutes sensitivity range 1010-15-15 - 10 - 10-9-9
If decay rate t If decay rate t 1/2 1/2 = 235 minutes sensitivity range = 235 minutes sensitivity range 1010-14-14 - 10 - 10-18-18
Prototype Device #1Prototype Device #1
BPAC December 2002 BPAC December 2002 Bacterial Required for Bacterial Required for
DetectionDetection
Bacillus cereusBacillus subtilisClostridium perfringensCornybacterium speciesEcherichia coliEnterobacter cloacaeKlebsiella oxytoca
Propionibacterium acnesPsudomonas aeruginosaSerratia marcesensStaphyloccocus aureusStaphylococcus epidermidisStreptococcus pyogenesStreptococcus viridans
Bacteria verified and Bacteria verified and validated for bead validated for bead
isolationisolation
AeromonasBacillusCampylobacterCitrobacterClostridiumE.coliEnterobacterEnterococcusHafniaKlebsiella
LactobacillusListeriaMorganellaProvidenciaProteusSalmonella(Serratia)ShewanellaShigellaVibrioYersinia
BordetellaBorreliaChlamydiaClostridiumCorynebacterium(E. coli)EnterobacterHaemophilusHelicobacterKlebsiellaListeria
MicrococcusMycobacteriumNeisseriaPropionebacteriumProteusPseudomonas(Salmonella)SerratiaStreptococcusStaphylococcusYersinia
GloBac™GloBac™
GloBac™GloBac™
Fully AutomatedFully Automated Touch Screen Touch Screen
DisplayDisplay Microprocessor Microprocessor
DrivenDriven Internal Reagent Internal Reagent
cartridgecartridge Top loading sample Top loading sample
portport
University of Minnesota University of Minnesota Field Evaluation TestingField Evaluation Testing
Dr. McCulloughDr. McCullough Dr. BowmanDr. Bowman Dr. Gundu Rao Dr. Gundu Rao Mary Clay: Project ManagerMary Clay: Project Manager Shelley Pulkrabek: Blood Bank CoordinatorShelley Pulkrabek: Blood Bank Coordinator Debbie Cocking Johnson: Related lab supportDebbie Cocking Johnson: Related lab support Nancy Ward: Technical SupervisorNancy Ward: Technical Supervisor
Bacterial Contam ination Detection in PlateletsUniversity of M innesota
N egative G lucose C onc entra te sto Pla te le t Pools
Pla te le t Poolsn=420 0
n=200 ne ga tive sto M ic ro la b
All N e ga tive s to T ransfusion
S BA + IDan d
Ba cT + ID
All Positives to M icro la b
G loBa c T estingn=420 0
N ega tive to Pla te le t Pools Positive to M ic ro lab
Positive or N e ga tivePerform G loBac
T estingn=21 ,000
Positives to M ic ro la b
G lucose M e te r testingn=21 ,000
Pla te le t C once ntra tesn=21 ,000
India StudyIndia Study
Bangalore Rotary Blood Bank StudyBangalore Rotary Blood Bank Study Expected contamination rate of over Expected contamination rate of over
6%6% Study size 2000 unitsStudy size 2000 units Expected positives greater than180Expected positives greater than180 Discussion with FDA on accepting Discussion with FDA on accepting
datadata
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