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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

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

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


– 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


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


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


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


A Stable Modular Design

Stable positive

ion portion

+

Variable mass

portion

Nucleotides

of primer

2

UV light

Mass post-cleavage = 729

June 7, 2011


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


Simultaneous Monitoring of 44 tagsDetection of 8 tags

June 7, 2011


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


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


MultiMaTCH

MassCode PCR products (n)(109)

Lambda exonuclease digestion

MassCode probe annealing and extension

Mass Tag Coded Hybrid

add admixture

June 7, 2011


Clean, Inject and Detect

UV light

Mass Tag Coded Hybrid

StrataPrep cleanup

Automated

Inject

Detect

June 7, 2011


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


Simultaneous Salmonella Detection and Subtyping

-500

500

1500

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3500

353

419

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431

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379

439

387

443

391

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487

403

459

407

463

411

467

479

471

Response -

Thre

shold

Dublin

-500

500

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2500

3500

353

419

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431

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379

439

387

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487

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Response −

Thre

shold

Saint Paul

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500

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4500

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Re

spo

nse -

Thre

sho

ld

Javiana

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500

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5500

353

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Response -

Thre

shold

Paratyphi A

No false positive calls or targets, only individual tagsNo false positive calls or targets, only individual tags

-500

500

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Response -

Thre

shold

Rubislaw

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2500353

419

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387

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Response -

Thre

shold

Poona

-500

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Response -

Thre

shold

Senftenberg

-500

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4500

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Re

spo

nse -

Thre

sho

ld

Newport

-500

500

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2500

3500

353

419

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375

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379

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387

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487

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463

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467

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Response -

Thre

shold

Agona

-500

500

1500

2500

3500

4500

353

419

357

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367

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431

375

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379

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387

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459

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Response -

Thre

shold

Montevideo

-500

500

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4500

5500

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Response -

Thre

shold

Enteritidis

-500

500

1500

2500

3500

4500

353

419

357

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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


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


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


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


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


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

14-PLEX PRIMERS: WHOLE GENOME BLASTN RESULTS

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

using masscode multiplexing agilent technologies, inc. · using masscode multiplexing agilent...

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