Detection of Bovine High Consequence Agents on an Electronic Microarray Platform
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Transcript of Detection of Bovine High Consequence Agents on an Electronic Microarray Platform
Detection of Bovine High Consequence Agents
on an Electronic Microarray Platform
Kimberley Burton HughesCanadian Food Inspection Agency
Lethbridge Laboratory
Bovine High Consequence Pathogen Assays
• Component 1: – Foot and Mouth Disease Virus
• Component 2: – FMD Differentials
• Vesicular Stomatitis• Exotic Malignant Catarrhal Fever • Rinderpest• Blue Tongue• BVD• BHV• Parapox complex
Clinical Symptoms:FeverBlisters in mouthExcessive salivationBlisters on feetWeight loss
FMD Strain Identification (with strain specific probes)
- Vaccine selection - Epidemiology
FMDPositive
+
Bovine HC Pathogen Assays: Overview Suspect case Detect and Serotype FMD
FMD ELECTRONIC MICROARRAY A
Ira
n 1
/96
A A
RG
2/2
00
1
A A
RG
87
A C
OL
/85
A2
2 I
ran
/99
A2
2 I
raq
24
/64
A2
4 C
ruze
iro
O1
Ma
nis
a
O T
AW
10
/97
O U
KG
11
/20
01
O1
BF
S 1
86
0
C1
No
ville
C3
Re
sen
de
AS
IA 1
PA
K 1
/54
AS
IA 1
Sh
am
ir
SA
T1
Ke
n 4
/98
SA
T1
Bo
t 1
/68
SA
T2
Zim
5/8
1
SA
T2
Sw
a 1
/69
SA
T2
Sa
u 1
/20
00
SA
T2
Zim
10
/91
SA
T3
Be
c 1
/65
SA
T3
Zim
4/8
1
A
B A
ASIA
SAT1
C
O
SAT2
SAT3
Neg Control
Ser
oty
pin
gp
rob
es
FMD Isolates
Neg Control
Detection probes
FMD ELECTRONIC MICROARRAY A
Ira
n 1
/96
A A
RG
2/2
00
1
A A
RG
87
A C
OL
/85
A2
2 I
ran
/99
A2
2 I
raq
24
/64
A2
4 C
ruze
iro
O1
Ma
nis
a
O T
AW
10
/97
O U
KG
11
/20
01
O1
BF
S 1
86
0
C1
No
ville
C3
Re
sen
de
AS
IA 1
PA
K 1
/54
AS
IA 1
Sh
am
ir
SA
T1
Ke
n 4
/98
SA
T1
Bo
t 1
/68
SA
T2
Zim
5/8
1
SA
T2
Sw
a 1
/69
SA
T2
Sa
u 1
/20
00
SA
T2
Zim
10
/91
SA
T3
Be
c 1
/65
SA
T3
Zim
4/8
1
A
B A
ASIA
SAT1
C
O
SAT2
SAT3
Neg Control
Ser
oty
pin
gp
rob
es
FMD Isolates
Neg Control
Detection probes
Heat map depicting fluorescent intensities of 2 detection (A) and 12 serotyping (B) probes against 23 isolates that represent all 7 serotypes.
Bovine HC Multiplex Testing for 7 FMD differentials
FMDNegative
FMD Strain ID Strategy
- 3 highly variable sites were identified within VP 1 coding region
- Probes were designed at the 3 sites for reference strains of each serotype
Reference strains selected: - WRLFMD recommended vaccine strains - major genetic lineages or topotypes (genetic lineages that fall within geographical boundries)
1 2 3VP 1
Example
Reference Strain
A B C D E F
Site 1
Site 2
Site 3
Unknown strain
Site 1
Site 2
Site 3
Referencestrains
Pro
bes
FMD O Strain Identification
*WRLFMD recommended vaccine strains**Samuel and Knowles, J. Gen Virol. 2001
Reference Strain Topotype**
*O Manisa Middle East - South Asia
*O TAW 10/97 Cathay
*O BFS 1860 Euro - South America
O GHA 5/93 West Africa
O MAL 98 East Africa - 2
O MYA 98 South-East Asia
Strains Tested
Pro
bes
Bovine High Consequence Pathogen Assays
• Component 1: – Foot and Mouth Disease Virus
• Component 2: – FMD Differentials
• Vesicular Stomatitis• Exotic Malignant Catarrhal Fever • Rinderpest• Blue Tongue• BVD• BHV• Parapox complex
Bovine HC Multiplex
PCR
BVD BHV VSV (NJ)
VSV (IND) RPV BTV
MC
FV
OR
F
BTV
Virus Amplicon(bp)
BVD 277
BHV ~432
VSV 270
RPV 235
MCFV 405
PPV 84
BTV 342
Entero 195
Ent
ero Clinical Neg Oral
NT
C
Bovine HC Electronic Microarray Results (PN)
Virus Strains
BVDV
BHV
VSV
RPV
MC
FV
BTV
OR
FV
En
tero
N
TC
1 2 NJ IND
Pro
bes
RPV Titration Series
0
2
4
6
8
10
12
14
RPV-P001 NDV HP VC22
Probes
Po
sit
ive
to N
egat
ive
Ra
tio
(P
N R
ati
o)
Sensitivity Bovine HC Assay
Virus PCR NC400
RP 0.1 0.1
VSV 2-3x103 2-3x103
BTV 100 100
BVD 31 31
BHV 68 6.8x102
Limit of Detection (TCID50/ml)103 102 101104 100 10-1 10-2 10-3
RPV 235 bp
TCID50/ml:
NC400 Results
Clinical Samples TestedVirus No. of
SamplesNo. of
AnimalsSample Type
VSV 24 2 NJ, 2 IND oral/nasal swabs
RPV 16 4 oral/nasal swabs
MCFV 24 2 Buffy coat
BTV 7 1 whole blood
BVD 6 (spiked) 3 oral/nasal swabs
BHV 4 (spiked) 2 oral/nasal swabs
Negative 36 31 oral/nasal swabs
VSV Oral Swabs Animal #855
0
10000
20000
30000
40000
50000
60000
70000
80000
BVDV Com BVDV1 BVDV2 IBR VSV RPV MCF PPV BTVa BTVb Entero Neg
Flu
ore
sc
en
t In
ten
sit
y
dpi -5
dpi 1
dpi 2
dpi 3
dpi 4
dpi 5
dpi 6
dpi 7
dpi 8
dpi 9
dpi 10
dpi 11
NTC
Pos
Neg
Strong, specific reaction starting at 2 dpi.
No cross reaction to other probes in assay.
Clinical Sample Testing Results
Advantages of electronic microarray: - highly multiplexed, type and subtype - robust platform - faster - higher throughput - simplifed data analysis
VSV Oral Swabs Animal #855
0
2
4
6
8
10
12
14
16
VSV Neg
Po
siti
ve:N
egat
ive
Rat
iodpi -5
dpi 1
dpi 2
dpi 3
dpi 4
dpi 5
dpi 6
dpi 7
dpi 8
dpi 9
dpi 10
dpi 11
NTC
PCR
NC400
RT-PCR*
DPI -5 1 2 3 4 5 6 7 8 9 10 11
Slide Array
- - + + + + + - - + - -
- - + + + + + - - + - -
- - - + - inc - - - inc - -
- - + + - + + - - + - -
* NCFAD completed RT-PCR
Current Work
• Adapt assays to new format– Fully automated, integrated system
• magnetic sample preparation• amplification• electronic microarray
– Portable, compact unit for on-site testing
– Fully bio-contained cartridge– Rapid– Low cost (~$10)
CFIA, LethbridgeOliver Lung Dirk Deregt Tara Furukawa-Stoffer Mathew FisherSamuel Ohene-AdjeiKristen Hahn*Anne Beeston*Jennifer Geddes*Billy Mauro*
Nexogen, Inc., San Diego, USA Dalibor Hodko
CFIA, NCFAD, Winnipeg John Pasick Yohannes BerhaneAlfonso Clavijo* Animal and Technical Staff
IAH, Pirbright, UKDonald KingScott Reid
Funding:
CRTI (The Chemical, Biological, Radiological or Nuclear Research and Technology Initiative)
* Former membersCFIA Lethbridge Laboratory
Bovine HC Multiplex PCR DevelopmentVirus Genome Gene
TargetAmplicon Size
Reference
Vesicular Stomatitis
-ssRNA L-gene (polymerase)
270 bp Lung et al. 2010(Unpublished)
Rinderpest -ssRNA F-gene(fusion protein)
235 bp Forsyth and Barrett 1995
MCFV dsDNA AlHV-1 ORF50(tegument protein)
405 bp Traul et al. 2005Li et al. 2000
Blue Tongue dsRNA Segment 1 (polymerase)
342 bp Toussaint et al. 2007 (modified)
BVD +ssRNA 5’ UTR 277 bp Deregt et al, 2006
IBR dsDNA gB 432 bp Adapted from Hindson et al. 2008
PPV dsDNA BL2 gene (envelope protein)
84 bp Nitsche et al. 2006
Enterovirus(Internal Control)
+ssRNA 5’UTR 195 bp Schwab et al. 1995
PN Values for heat mapUNQID NAME New York 1Singer Hastings 24515 890 Ames-125cColorado #34V654 V135 #5 Edmonton#39 EdmontonV76 NJ 0185 PNBNJ 0804COE1NJ 1184 HDBNJ 89 GASNJ 92 CLB NJ New Mexico 95IN 85 CLB IN 94 GUB IN 97 CRB IN 98 COE IN ATCC IN 01V1008Kabete O Eldoret Buffalo PakchongWC11 NZ2 Serotype 2 Serotype 8 Serotype 10Serotype 11Serotype 12Serotype 13Serotype 14Serotype 17Entero NTCBVDcom BVDcom 11.76577 7.754208 7.804652 9.714948 3.256222 10.80851 1.35928 1.504666 1.239458 1.622801 1.272246 1.331403 0.812308 1.0339 1.118109 0.970975 1.080153 0.863726 0.851554 0.99503 0.985961 1.195056 0.830047 0.836354 1.187438 1.110822 1.079357 1.169616 7.526438 0.890285 1.117507 0.977176 1.197821 1.189438 1.157167 1.09731 1.171655 1.442352 1BVDV 1 BVDV 1 8.091065 5.345374 6.269013 1.604339 1.028921 1.043356 1.236615 1.314306 1.14814 1.271472 1.20234 1.359998 0.834306 0.943152 0.850309 0.84251 0.978906 0.815397 0.948131 0.986479 1.020446 1.036417 0.849969 0.785233 1.062698 0.992673 0.999221 1.035766 1.123631 0.775337 0.865609 0.918671 1.008658 0.847118 1.020732 1.015398 0.973768 0.990738 1BVDV 2 BVDV 2 1.192644 1.480582 1.0721 4.105763 2.739955 2.324881 1.582017 1.274558 1.205 1.368776 1.372848 1.520939 0.788122 1.147097 0.967384 0.89655 0.918711 0.816881 0.978715 0.894517 1.039074 1.04704 0.820648 0.818492 1.103149 1.034101 0.994347 1.00778 4.092187 0.734854 0.98943 0.976453 1.078531 0.980549 1.01028 0.949754 0.954165 1.002302 1IBR IBR 1.17609 1.252557 1.312911 1.349983 1.359995 1.36139 3.748538 3.589743 3.430233 3.080304 2.624018 2.885781 0.858442 1.030387 1.036894 0.924783 1.015739 0.838732 0.97224 1.034032 1.044263 1.108854 0.870049 0.835017 1.244105 1.061456 0.976465 0.757239 1.438995 0.935944 1.031932 1.112509 1.201936 1.19101 1.233181 1.196953 1.181384 1.25162 1VSV VSV 0.990311 1.340782 1.515463 1.540198 1.380635 1.478721 1.291367 1.813942 1.691816 1.596399 1.256628 1.051135 12.93494 13.58493 5.493361 12.47535 14.00174 13.1101 14.41345 14.32443 12.42949 12.53508 11.66703 12.35545 1.21965 1.259974 1.291757 0.951098 1.169733 0.576439 0.937514 0.852491 0.998242 0.916786 0.942873 0.926176 0.906602 0.90579 1RPV RPV 0.813762 0.850913 0.863405 0.82887 0.879974 0.857929 1.360662 0.7923 0.892522 0.847839 0.935839 0.946198 0.63565 0.803239 0.799678 0.765563 0.931487 0.793653 1.093582 1.026186 0.929206 1.031193 0.672809 0.816834 7.878836 5.648224 6.249232 0.977972 1.061381 0.734397 0.907609 0.929925 0.989575 0.972016 0.952307 0.907815 0.963 0.953902 1MCFV MCFV 1.083654 0.975525 0.961446 1.028907 1.053073 1.185808 1.179411 1.306567 1.144135 1.340048 1.309899 1.281784 0.784625 1.025706 1.009291 0.936185 0.940468 0.817093 0.992365 1.03337 0.994761 1.136021 0.807855 0.830696 1.145384 1.051836 0.936567 3.188325 0.737744 0.722477 0.833866 0.853611 0.899973 0.872598 0.89973 0.904135 0.968358 0.790019 1PPV PPV 1.289462 1.015418 1.235608 1.142404 1.00408 1.10624 1.192285 1.151036 1.198627 1.54175 1.333828 1.400519 0.594427 0.965978 0.944656 0.864962 1.023453 0.882464 1.062199 1.037955 1.088784 1.153146 0.906359 0.868475 1.181598 1.074598 0.954886 0.991382 9.351835 0.505999 0.851102 0.856463 0.889961 0.880337 0.917765 0.884074 0.919851 1.981599 1BTVa BTVa 1.255229 1.220215 1.260638 1.258259 1.18539 1.181823 1.901321 1.527834 1.286354 1.50709 1.462994 1.646308 0.973744 1.069918 0.958822 0.887444 1.052105 0.832121 1.113666 1.083519 1.03506 1.194128 0.96097 0.887396 0.690606 1.077383 1.083188 0.939982 1.170573 13.44441 12.1667 12.78084 12.54584 12.98281 13.00731 10.98303 13.35975 1.434924 1BTVb BTVb 1.104666 1.076057 1.091841 1.098475 1.059821 1.008122 1.126359 0.946015 1.019326 1.26406 1.257478 1.410889 0.799105 1.005829 0.956727 0.903884 0.967964 0.757045 0.958591 0.941938 1.006405 1.101974 0.793738 0.78154 1.049887 0.954058 0.95672 0.951752 1.175902 0.874435 1.218597 1.137489 1.14816 0.954699 0.872045 1.013138 0.938476 0.989955 1Entero Entero 0.717104 1.126007 1.190686 1.215852 1.223931 1.262037 1.191166 1.261282 1.188318 0.995047 1.186688 1.131555 0.8368 1.045319 0.959019 0.90408 1.036192 0.840817 0.902673 0.852565 0.992191 1.085978 0.836666 0.823407 1.052034 1.006911 1.018597 1.129746 1.199476 0.851214 1.020737 1.075995 0.984253 1.046272 1.108857 1.029543 1.023077 2.531845 1Neg ControlNeg Control 1.16786 1.19656 1.251484 1.189663 1.229557 1.2729 1.249676 1.477487 1.320412 1.240144 1.422093 1.485707 1.030243 1.257607 1.184943 1.090684 1.24808 0.986837 1.309383 1.174262 1.103398 1.319519 0.941855 0.857224 1.23534 0.941093 0.908428 0.853934 0.995909 0.742915 0.948758 0.818555 0.985439 0.956848 0.935025 0.911065 0.82454 0.89869 1
0
5000
10000
15000
20000
A-T-23 A-T-27 C24 O148 mod1 O-T-16 O-T-8deg ASIA-208mod2
SAT1-215mod3
SAT2-T-23deg
SAT2-T-31 SAT2-T-9deg
SAT3-9 Neg. Cont.Probes
FI
A Iran 1/96 A ARG 2/2001A ARG /87 A COL/85A 22 Iran/99A 22 Iraq 24/64A 24 CruzeioO 1 Manisa O TAW 10/97 O UKG 11/2001 O 1 BFS /860C 1 Noville C 3 Resende ASIA 1 Pak 1/54ASIA 1 ShamirSAT1 Ken 4/98SAT1 Bot 1/68SAT2 Zim 5/81SAT2 Swa 1/69SAT2 Sau 1/2000SAT2 Zim 10/91SAT3 Bec 1/65SAT3 Zim 4/81
Probes
FMD Strain Identification (with strain specific probes)
- Vaccine selection - Epidemiology
FMDPositive+
Bovine HC Pathogen Assays: Overview
Sample from suspect FMD case
Simultaneous detection and serotyping of FMD
FMDNegative
Bovine HC assay Testing for 7 other HC agents
and differentials
-
Foot and Mouth Disease Virus
– Detection (polymerase gene)– Serotyping (VP3 gene)– Strain Identification
Bovine High Consequence Agents
Previously presented
Foot-and-Mouth Disease
• 7 serotypes: A, O, C, Asia 1, SAT 1, 2, 3
• Confirmation requires laboratory diagnosis
• Highly infectious, expensive to control, and economically devastating
• Last cases in Canada:1953 (US 1929)
Clinical Signs:FeverBlisters in mouthExcessive salivationBlisters on feetWeightloss
Current Work
New “Sample-to-Answer” Technology
• Fully Automated Molecular Detection
• Integrated Sample Preparation and DNA Electronic MicroarrayDetection
•Rapid
•Low cost
Portable I nstrument Disposable cartridge
Compact Bio-Contained
New “Sample-to-Answer” Technology
• Fully Automated Molecular Detection
• Integrated Sample Preparation and DNA Electronic MicroarrayDetection
•Rapid
•Low cost
Portable I nstrument Disposable cartridge
Compact Bio-Contained
Rinderpest Oral Swabs
0
5000
10000
15000
20000
25000
30000
35000
40000
RPV MCF VSV BTVa BTVb BVDV Com BVDV1 BVDV2 IBR PPV Entero Neg Control
Flu
ore
sc
en
t In
ten
sit
y
C430 dpi 0
C430 dpi 6
C1035 dpi 0
C1035 dpi 6
NTC
Positive Control Pool
Negative Control
DPI
Rinderpest Oral Swabs
0
1
2
3
4
5
6
7
8
RPV MCF VSV BTVa BTVb BVDV Com BVDV1 BVDV2 IBR PPV Entero Neg Control
Po
sit
ive
:Ne
ga
tiv
e R
ati
o
C430 dpi 0
C430 dpi 6
C1035 dpi 0
C1035 dpi 6
NTC
1. RNA isolation
2. RT-PCR amplification of target
++
Positive Charge
++
Positive Charge
Printing
Biotinylated Capture Probe
Amplicon
Hybridization
Wash
Reporting
NC400
1. Print biotinylated capture probes
2. Add amplicon
Biotinylated capture probe
Amplicon
reporter probe
3. Add reporter probe
4. Measure fluorescence at test site
Electronic Microarray• Electrophoretically driven printing and hybridization
Time (seconds) Time (hours) Electronic Passive
(Movie here)
CMOS Chip PlatinumMicroelectrode
Hydrogel Permeation LayerContaining Streptavidin
50 m diameter test site
Array
• 400 independently activated test sites
1. RNA isolation
2. RT-PCR amplification of target
++
Positive Charge
++
Positive Charge
Printing
Biotinylated Capture Probe
Amplicon
Hybridization
Wash
Reporting
Biotinylated capture probe
NC400
1. Print biotinylated capture probesAmplicon
2. Add amplicon
reporter probe
3. Add reporter probe
4. Measure fluorescence at test site