Using MassCode Multiplexing Agilent Technologies, Inc
Transcript of Using MassCode Multiplexing Agilent Technologies, Inc
MultiMaTCH Genotyping A Novel Rapid Method for the
Simultaneous Detection and
DNA Subtyping of Salmonella
Using MassCode Multiplexing
Agilent Technologies, Inc.Santa Clara, California
Lenore Kelly, Ph.D.
Americas Food Biochemist
SSAOAC
Trace-Back: a Situational Analysis
•Serovar typing is a lengthy procedure with turnaround times
exceeding the half-life of a typical outbreak
•Sampling during an outbreak is increased an order of
magnitude over steady state monitoring
•Outbreaks are increasing in frequency due to globalization of
the food supply
• Rapid detection methods desired:
– Faster identification of problems
– Better protection of public health
– Quicker release of non-infected products
June 7, 20112
Page 3
Why is Salmonella Serotyping Important?
• Serves as the basis for the National Salmonella Surveillance system
• “International language” of Salmonella
• 60+ Years of surveillance data based on serotype
• Approximately 40,000 isolates serotyped each year by state health
departments and federal agencies.
• Critical for epidemiologic classification of strains and for outbreak
investigations
Agilent Confidential
June 7, 2011
Page 4
Disadvantages of Traditional Salmonella Serotyping
• Requires >250 antisera to identify all serotypes
• Production and QC with the antisera is problematic.
• Requires >350 strains and antigens to maintain sera.
• Takes a minimum of 3 days to identify all antigens of an isolate
Agilent Confidential
June 7, 2011
Project Aims:
Combine 2 of 3 steps
Faster response: ≤ 30 h
More informative results
June 7, 2011
Emerging Technologies in Times of Change
Salmonella Characterization
DetectionGenus, species
qPCR, etc 8-30 h
Traditional: 3-5 days
Serotyping
Serology: 4-5 days
Strain typing
PFGE,MLST, etc.:
5-10 days
Subtyping
Serogroup Serovar Genovar
– challenging, > 2,500 serovars
– paucity of genome sequence information = difficult molecular subtyping
Genomes
Discrimination and Information
SensitivitySpecificity
Surveillance
Outbreak response
Outbreak investigation
Aim of Our Applied Research
Reliable detection and molecular typing of foodborne pathogens
using a single PCR-based assay
June 7, 2011
Emerging Technologies in Times of Change
– novel labeling technology allowing multiplexed detection (10-40plex)
– probe format
– compatible with real-world sample matrices
– software package allowing simple instrument control and automated
results analysis
Current solution with a working prototype
MultiMaTCH methodology with MassCode technology
June 7, 2011
Emerging Technologies in Times of Change
MultiMaTCH System Workflow
MultiMaTCH
w/MassCode
PCR
StrataPrep
cleanup
extract gDNAprimary enrichment
10-40plex
96 samples
6 h post enrichment
detect
Reagents, consumables, instrumentation and software provided by Agilent
356
MassCode TagsA Novel Reporter for Biomolecules
– MassCode labels give biomolecules a digital code
June 7, 2011
Emerging Technologies in Times of Change
370 374366356352 378 382 T390386 394 398
422 434426 470466 478 482T450 462458454446442438430
T 518506 514486 494 498 502 510 534 538530526522 543 547
T589 601593 617613
418414402 406 410
474
542
609605597573569 577 581 585565561557
621 625 633629 645 649641637 T657653 661 665 685669 677673
T693 705701697689 733713 717 721709 725 729
Daltons
Dalto
ns
– 93 unique tags = high density multiplexing
– A true liquid array: Solution based; No beads; No solid supports
June 7, 2011
Emerging Technologies in Times of Change
A Stable Modular Design MassCode tagged primer
Mass post-cleavage = 356
Stable positive
ion portion
+
Variable mass
portion
5Nucleotide
s of primer
Oligos are synthesized by Operon with a 6-amino-1-hexanol linker on the 5‟-terminal phosphate.
The 6-amino- group is covalently coupled to a photocleavable MassCode tag.
UV light
June 7, 2011
Emerging Technologies in Times of Change
A Stable Modular Design
Stable positive
ion portion
+
Variable mass
portion
Nucleotides
of primer
2
UV light
Mass post-cleavage = 729
June 7, 2011
Emerging Technologies in Times of Change
Discrete Resolution of 93 tags
Selective Ion Monitoring of tags
15.5 in
26 in
29 in
Benchtop instrument
No spectral overlap
June 7, 2011
Emerging Technologies in Times of Change
Simultaneous Monitoring of 44 tagsDetection of 8 tags
June 7, 2011
Emerging Technologies in Times of Change
MassCode PCRA Multiplex PCR
Foodborne Pathogen PanelMassCode tag
Target organism F primer R primer
Campylobacter species
Campylobacter jejuni
Campylobacter coli
E. coli species
E. coli O157:H7
non-O157 STEC
ETEC
Vibrio cholerae
Vibrio parahaemolyticus
Vibrio vulnificus
Staphylococcus aureus
Enterobacter sakazakii
Salmonella species
Salmonella enterica Typhimurium
Salmonella enterica Enteritidis
Listeria species
Listeria monocytogenes
Shigella species
Yersinia enterocolitica
Internal Inhibition Control
374
356
382
426
446
438
506
514
494
498
573
581
565
561
625
645
637
T693
705
713
370
352
378
422
434
442
T486
502
510
569
577
557
621
633
629
641
701
697
689
709
1 well/sample
20 targets/well
Flexible assay design
Dual reporting/target (QC)
High throughput
MultiMaTCHMethod for the Multiplexed Synthesis of Mass Tag Coded Hybrids
June 7, 2011
Emerging Technologies in Times of Change
A one-step temperature dependent reaction
Probe detects correctly amplified targets from MassCode PCR
Greater assay specificity
Addition of admixture directly to MassCode PCR reaction tube
Probe uniquely labeled with MassCode tag
Retains dual reporting system
June 7, 2011
Emerging Technologies in Times of Change
MultiMaTCH
MassCode PCR products (n)(109)
Lambda exonuclease digestion
MassCode probe annealing and extension
Mass Tag Coded Hybrid
add admixture
June 7, 2011
Emerging Technologies in Times of Change
Clean, Inject and Detect
UV light
Mass Tag Coded Hybrid
StrataPrep cleanup
Automated
Inject
Detect
June 7, 2011
Emerging Technologies in Times of Change
Salmonella MultiMaTCH Assay DesignA 14plex Hierarchical Subtyping Assay
Typhimurium
394 T450
398 487
Agona
462406
Dublin
470478
Paratyphi A
466410
Typhi
366 426
B
370 430
C1
374 434
C2
378 438
D1/A
386 442
E1
446T390
G
356 422
Salmonella
352 418
IAC
458402
Enteritidis
SerovarSerogroup
Proof-of-concept prototype
Exclusion panel (so far)
• E. coli O157:H7
• E. coli species
• Bacillus subtilis
• Human
• Campylobacter jejuni
• Listeria monocytogenes
Allele-specific signatures
June 7, 2011
Emerging Technologies in Times of Change
Simultaneous Salmonella Detection and Subtyping
-500
500
1500
2500
3500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Response -
Thre
shold
Dublin
-500
500
1500
2500
3500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Response −
Thre
shold
Saint Paul
-500
500
1500
2500
3500
4500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Re
spo
nse -
Thre
sho
ld
Javiana
-500
500
1500
2500
3500
4500
5500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Response -
Thre
shold
Paratyphi A
No false positive calls or targets, only individual tagsNo false positive calls or targets, only individual tags
-500
500
1500
2500
3500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Response -
Thre
shold
Rubislaw
-500
500
1500
2500353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Response -
Thre
shold
Poona
-500
500
1500
2500
3500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Response -
Thre
shold
Senftenberg
-500
500
1500
2500
3500
4500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Re
spo
nse -
Thre
sho
ld
Newport
-500
500
1500
2500
3500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Response -
Thre
shold
Agona
-500
500
1500
2500
3500
4500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Response -
Thre
shold
Montevideo
-500
500
1500
2500
3500
4500
5500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Response -
Thre
shold
Enteritidis
-500
500
1500
2500
3500
4500
353
419
357
423
367
427
371
431
375
435
379
439
387
443
391
447
395
451
399
487
403
459
407
463
411
467
479
471
Res
po
nse
− T
hre
sho
ld
Typhimurium LT2
Serovar
Serogroup
error bars = STDEV calculated from 3 biological replicates
individually run in 3 different experiments over a 13 day period
June 7, 2011
Emerging Technologies in Times of Change
Sensitivity of the Salmonella MultiMaTCH AssayPreliminary Results from 3 Isolates
Serovar [SGSC]
# Targets
Amplified
DNA Copies
Detected per Target
Rubislaw [2511] 1 219
Newport [4910] 2 1,450
Enteritidis [4901] 3 1,830
Determined by using gDNA extracted from cells cultured in BHI
Decrease in sensitivity as # of detected targets increases
This tradeoff provides greater subtyping capabilities in a short amount of time
Right amount of discrimination for rapid response to outbreaks
and surveillance?Discrimination and Information
Sensitivity Specificity
Surveillance
Outbreak response
Outbreak investigation
June 7, 2011
Emerging Technologies in Times of Change
Detecting Enteritidis Contamination of a Tomato
Salmonella
MultiMaTCH
StrataPrep
cleanup
DNA template preparation with Instagene matrixSpike 0.75 CFU/g, enrich 14 h
detect
Serovar
Serogroup
June 7, 2011
Emerging Technologies in Times of Change
Robust System Design
Multiple targets per subtype promotes very low
type 1 error rateTwo tags per target
IAC
Safeguard mechanisms built into assay
Systems and Assay Controls
NTC
External positive control
External negative control
performed in the
same well
Instrument integrity control
reduces type 2 errors
Automated instrument calibration
June 7, 2011
Emerging Technologies in Times of Change
Advantages of the MultiMaTCH System
Multiplexing allows subtyping – 93 tags (not limited to 4 or 5 dyes)
All hybridizations are solution-based
Cost-effective
Rapid method - 6h
Less PCR competition, all amplicons similar in size
Automated data acquisition and analysis
High throughput – a 25plex = 2,400 individual tests for 96-well
Dual reporting per target – built in QC
Greater specificity than PCR alone
Flexible assay design
Not sensitive to ambient light
June 7, 2011
Emerging Technologies in Times of Change
Foodborne Pathogen Project team in Agilent Labs
Lead R&D Scientist
Gregory Richmond
R&D Scientist
Dan Ryan
Research Associates
Htet Khine
Tina Zhou
Software
Tony Brand
Managers
Kevin Killeen
Mary McBride
Hongfeng Yin
Life Sciences Group
Yves Konigshofer
Dorothy Yang
Executive Support
Neil Cook
Darlene Solomon
Richmond, G.S., Khine, H., Zhou, T.T., Ryan, D.E., Brand, T., McBride, M. T.,
Killeen, K. MassCode Liquid Arrays as a Tool for Multiplexed High-
Throughput Genetic Profiling. PLoS One, 6: e18967
Agilent Technologies collaboration with California
Animal Health and Food Safety
Purpose of collaboration
• Redesign several of the primers, testing amplicons in singleplex
• Validate the 14-plex assay with real samples
• Test specificity against library of pathogen strains
• Develop vertical and horizontal multiplex panels for multiple food
pathogens and for Salmonella serovars
MULTIPLEX PCR: WHAT’S NEXT?
Identification of new targets for serovars
Typhirumium, Agona, Typhi, and Paratyphi A
is in progress
What’s available?
seven complete genomes for Typhirumium,
two complete genomes each for Typhi and Paratyphi,
and a single complete genome for Agona
Access to more than one complete genome for each
serotype will be very helpful for the identification of
new targets
How does having more than one complete genome help?
This is a whole genome alignment of six Typhimurim
genomes using MAUVE
It clearly shows that the genomic architecture is highly conserved,
and a small genomic island is present in three strains (green block)
How does having more than one complete genome help?
This is a whole genome alignment of seven different strains/serovars
of S.e.e. using MAUVE
It shows where the genomic architecture is conserved, and
where it varies: there are definitely some genomic hotspots that are
prone for variation between strains/serotypes
The first half of the table is a whole genome comparison of Typhimurium
(LT2) with Newport, Heidelberg, Enteritidis, Dublin, Choleraesuis, and Agona
using RAST:
„Orthologs‟ are genes that are in common, and „specific CDS‟ are genes that
are found only strain LT2
Dr. Weimer‟s lab has performed CGH experiments for 11 serotypes using a
Affymetrix chip that contains LT2 genes. Analysis of this data is in progress
Comparative genomics and CGH analyses of
Salmonella enterica subspecies enterica strains