Multiplex Molecular Diagnostics – Principles for Shifting the Molecular

Diagnostics Paradigm

David L. Dolinger, Ph.D. Seegene, Inc.

Background Information

• Approximately ~ 80% of all clinical decisions are guide by diagnostics tests

–  Diagnostic tests account for 2 - 8% of ALL medical costs

• On average, in the developed world, 30% of infectious diseases (parasitic, viral, fungal and bacterial) go undiagnosed or misdiagnosed

–  Examples

•  Diarrhea – up to 50%

•  CDI – up to 60% for one study

–  What is the burden to the healthcare system?

–  What is the burden to the patient?

Molecular Diagnostics (MoDx)

•  Paradigm –  One sample – One analyte – One result –  Lack of sensitivity or specificity –  Target drift –  Turn around times –  High to moderate complexity –  Cost –  Tests that are Confirmatory –  Provide minimal

Actionable Information –  No active vigilance system –  Previously unidentified pathogens –  Lack of understanding of disease

syndromes

•  Result – Treatment decisions empirically based –  Leads to:

•  Inappropriate treatment •  Misuse of antibiotics •  Multi-drug resistance organisms

•  Outbreaks •  Morbidity and Mortality

MoDx – Current Paradigm

MoDx – Current Paradigm

What could you do with an Assay that could ask Twenty Questions,

an Assay that is a Diagnostic Assay?

What is Required for a Paradigm Shift

•  What is required for such an assay? –  Performance?

•  Sensitivity •  Specificity •  Reproducibility

•  Accuracy

–  Multiplexity? •  Number of Analytes

•  Number and type of Controls

–  Built-in Quality Assurance? –  Sample?

•  Types •  Pre-analytical steps

–  Cost? •  Versus singleplex •  Versus multiple singleplexs’

•  One Assay – One Analyte

•  One Channel – One Target

•  Controls

•  Non-specific Oligonucleotide Binding

•  Primer-dimer Formation

•  Sensitivity/Specificity indirectly related

•  Assay Optimization and Balance

•  Assay Quality Assurance

Hurdles in the Development of NexGen MoDxs’

Principle of DPO™

Template DNA 5’

3’ 5’

Target site

DPO™ (Dual Priming Oligonucleotide) Polydeoxyinosine Linker

3’

•  How to increase specificity with out changing the basic thermodynamics and kinetics of PCR? •  Increased primer length increases specificity •  BUT increased primer length increases the Tm

“First priming” The longer 5’-portion preferentially

binds to the template DNA and initiates “stable

annealing”

5’ 3’ 5’

3’ X

“Second priming” The short 3’-portion selectively binds

to the target site and allows extension to occur

Target site 5’

Extension 3’

5’ 3’ O

Principle of DPO™

Target site Event 1

Event 2

Non-target site

Non-target site

3’

5’ No extension

3’ 5’ X

Although the longer 5’-portion binds a non-target site, due to thermodynamics the 3’-portion resists non-specific binding effectively block elongation

5’ No annealing

3’

5’ 3’ X

Due to thermodynamic constraints the 3’-portion alone fails to bind the target at an annealing temperature.

Built-in Quality Assurance

Process

DPO™ - Overcoming Hurdles of Multiplex PCR

•  Issues •  Primer dimers and other oligo – oligo interactions •  Non-specific productive binding •  Sensitivity and Specificity inversely related •  Complex assay optimization and balancing

•  Solution provided by DPO™ primers •  Dual-specificity priming – built in quality assurance •  NO non-specific productive binding •  NO primer dimer formation •  High Sensitivity [Low to NO False Negatives] •  High Specificity [Low to NO False Positives] •  As sensitive as a singleplex when other targets present •  Thermodynamics of ‘normal’ primers •  Kinetics of ‘normal’ primers •  Sensitivity and Specificity directly related •  Tolerant chemistry – easy optimization

Challenges Solutions

DPO™

One Assay – One Analytes √

One Channel – One Target

Controls

Non-specific oligonucleotide binding √

Primer-dimer Formation √

Sensitivity/Specificity √

Optimization and Assay Balance √

Quality Assurance √

NexGen MoDx Solutions

Tagging portion

Predetermined and Controlled Tm value

5’

5’

5’

3’

3’

3’

3’ 5’

3’ 5’

3’ 5’

5’

3’

5’

3’

5’

3’ 3’

Principles of TOCE™

Template (Serial dilution)

TOCE-based multiplex Monoplex (TaqMan)

Repeat 1 Repeat 2 Repeat 1 Repeat 2

10-1 32.10 32.00 31.90 31.91

10-2 36.30 36.66 35.10 35.36

10-3 39.38 40.03 40.08 41.63

10-4 ND ND ND ND

NC ND ND ND ND

A. TOCE-based multiplex (9-plex) B. Singleplex (TaqMan)

① ② ③

④

Performance equivalent to singleplex reactions

TOCE™ - Sensitivity

① ② ③

④

A. Target : Extension & Signal

B. Non-target : No extension & No signal

-○- Perfect match

-△- Mismatch (1-mer)

-X- Mismatch (2-mer)

-□- Mismatch (3-mer)

5’

5’

5’

3’

3’

3’

5’

5’

3’

3’

3’

3’

5’

5’

TOCE™ - Specificity

R2=0.999

Copy Number 107 106 105 104 103 102 101

Ct value 18.02 21.39 24.70 27.91 31.36 34.64 38.79

Reaction Linearity

107 106 105 104 103 102 101

TOCE™ - Performance Characteristics

TOCE™ - Detection (Screen) & Differentiation

TOCE technology is able to screen(A) and identify(B) multiple pathogens simultaneously in a single fluorescence channel. Tm difference between peaks is pre-determined, which enables to simultaneously detect minor species in mixed background containing major species.

Detection of Parainfluenza Differentiation of Parainfluenza 1 & 2 & 3

Cyclic Melting Curve Analysis (CMCA):

A method for analyzing melting curve repeatedly during the PCR process

30 cycles 40 cycles 50 cycles

Multiple Quantification in a Single Channel

Sample CMCA points

Results 30 cycles 40 cycles 50 cycles

1

A: >104

B : <101

C: 101~104

2

A: Negative

B: 101~104

C: <101

Standard (copies) >104 101~104 <101

A B C

A B C

Example of triple quantification in a single channel

60°C 68°C 75°C

60°C 68°C 75°C

* A, B and C indicate three different targets.

A B C

60°C 68°C 75°C 60°C 68°C 75°C

A B C

60°C 68°C 75°C

A B C

60°C 68°C 75°C

TOCE™ - CMCA

Mutant Target

Wild-type Target

Mutation site

Mutant-specific

Mutant-specific

TOCE™ - Mutational Analysis

59 71 65 77°C 59 71 65 77°C

G A

C T

(Sequence alignment of 16s rRNA genes)

Mycobacterium avium complex Mycobacterium abscessus Mycobacterium kansasii

A G G T T C

Mycobacterium avium complex

Mycobacterium abscessus

Mycobacterium kansasii

GTGGGATGGGCCCGCG************TTGGTGGGGTGATGGCCTACCAAGGCGAC

GTGGGATGAGCCCGCG************TTGGTGGGGTGATGGCCTACCAAGGCGAC

GTGGGATGGGCCCGCG************TTGGTGGGGTGACGGCCTACCAAGGCGAC

Single base specific Pitchers

59 71 65 77°C

TOCE™ - Single Base Discrimination

Example of Application to NTM Speciation

Challenges Solutions

DPO™ TOCE™

One Assay – One Analytes √ √

One Channel – One Target √ √

Controls √ √

Non-specific oligonucleotide binding √

Primer-dimer Formation √

Sensitivity/Specificity √

Optimization and Assay Balance √ √

Quality Assurance √ √

NexGen MoDx Solutions

A New Concept of real-time PCR

Current real-time PCR TOCE based real-time PCR

Technology & Principle

Design of Labeled Probes

No. of target 1 5 … 10 … N 1 5 … 10 … N

No. of required labeled probe

1 5 … 10 … N 1 1 1 1 1 1

Amount of each labeled probe 1 X for each probe 0.1 Х for each probe

Price of labeled probe (n=N)

$ ( 1 x N )

e.g.) $ 200 X ( 1 x 10 ) = $ 2,000

$ ( 0.1 x 1 )

e.g.) $ 200 X ( 0. 1 x 1 ) = $ 20

Concept To detect given number of targets, labeled probe should be designed and produced for eac

h target

Common labeled probe can be universally used for each target, saving time, labor, and prod

uction cost significantly

“Labeled universal probe” “Target specific labeled probe”

Key Features and Benefits of DPO™/TOCE™

Feature/Differentiator Benefit

Flexible and Agile

Rapid primer design and assay development; minimal redesign required Ease of development due to robustness of the chemistry Sensitivity is not sacrificed for specificity Built-in quality check due to dual priming events being required for each primer

Multiplexing Capabilities

Low spurious cross-reactivity ensures that most acquired targets can be tested

Minimal to no oligonucleotide interactions (primer dimers, complex secondary structures) simplify primer design and increases

multiplexing capabilities

Analytical and Clinical Performance

High Specificity ensures a low-to-no False Negative Rate, increasing confidence in clinical determination High Sensitivity enables lower detection limits and ensures low-to-no False

Positive Rate Quality Control built in to the reaction due to the characteristics of

DPO and TOCE

Key Features and Benefits of DPO™/TOCE™

Feature/Differentiator   Benefit  

Chemistry

Freedom from target sequences – No unambiguous results Controllable Tm Readout – repeatable data results Robust – tolerance to wide range of temperatures High Specificity – No False Positives High Sensitivity – No False Negatives Detection and Differentiation – Automatable – due to robust chemistry; homogenous reaction Adaptable – portable to multiple instrument platforms Quality Control – built into the chemistry low to NO false results

Multiplex Reaction

Homogenous Reaction – easily automatable Co-infection – determine multiple infections with the sensitivity of singleplexs Syndrome Based – panel based diagnostics

Multiplex Quantification Quantification – linear for dynamic range Repeated Tm Analysis – allows for setting of cutoffs and quantification Cut-off – when is infection actionable

Multiplex Mutational Analysis Single base discrimination – actionable information Insertion/Deletion – actionable information

DPO™/TOCE™ Technology Advantages

•  DPO™ –  Quality check – low to no False Positives or False Negatives

•  Four binding events required for a positive result

–  No decrease in performance, multiplex versus singleplex •  High Specificity •  High Sensitivity

•  TOCE™ –  Quality check – low to no False Positive or False Negatives

•  Multiple unique binding events required •  One specific cleavage event required

–  No decrease in performance, multiplex versus singleplex •  High Specificity •  High Sensitivity

–  Decreased cost of goods •  Decreased quantity of probes •  Minimal increases in enzymes versus singleplex

What is the Paradigm Shift

•  Freedom –  Develop

•  Assays that are intelligently designed –  Syndrome Based –  Detect and Differentiate –  Semi-quantitative or quantitative –  Provide Actionable Information –  Homogenous High multiplex Assays

–  Solutions •  Impact Patient Care for the better •  Provide Actionable Information that affects outcome •  Consider Healthcare Economics •  ‘True’ Diagnostic in their use and utility

One Sample – One Tube – Multiple Results

A Quantum Leap in MoDx

High Multiplex Target Detection and Differentiation in real-time

High Multiplex Target Quantification in real-time

High Multiplex Point Mutation Detection in real-time

TOCE™ - Melting Temperature Analysis (MTA)

TOCE™ - cyclic capture Melting Temperature Analysis (cyclic-CMTA)

TOCE™- Cleavage Site Specific Extension (CSSE)

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