TOCE™ Technology

The New Paradigm for High multiplex Real-time PCR

Outline of the technology

High multiplex Real-time Quantitative PCR

TOCE™ technology enables confirmation of multiple (five or more) target genetic detection and genetic variation. TOCE™ can implement qualitative test that tests multiple clinical samples and quantitative analysis bases on Cyclic Catcher Melting Temperature Analysis (cyclic-CMTA).
The profile of information generated by TOCE™-based high multiplex PCR analysis can substantially accelerate informed accurate diagnosis and treatment . As such, TOCE™ -based molecular testing is causing a paradigm shift in molecular diagnostics toward the high-content/high-quality testing needed for optimized patient treatment.


Technical Papers

Seegene Bulletin - VOLUME 1 JULY 2012

THE SCIENCE AND BUSINESS OF MOLECULAR DIAGNOSTICS

  1. High Multiplex Molecular Diagnostics - Shifting the Diagnostics Paradigm Jong-Yoon Chun. Ph D
  2. TOCE : Innovative Technology for High Multiplex Real-time PCR Dae-Hoon Lee, Ph.D.
  3. cyclic-CMTA : An Innovative Concept in Multiplex Quantification In-Taek Hwang, Ph.D.
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Features of the technology

1. Principles of TOCE™ technology

The key components for TOCE™ technology are DPO™ primer pairs, Pitchers and Catchers. The DPO™ is a Seegene's proprietary target-specific primer and provides highly specific amplification of the target region. The Pitcher is a tagging oligonucleotide which hybridizes specifically to the target region. The Catcher is a fluorescently labeled artificial template.

Principles of TOCE™ technology

2. High multiplicity in a single channel using Catcher -Tm analysis

TOCE™ technology enables identification of multiple target analytes simultaneously in a single channel. The signal can be measured in real-time and/or analyzed by Catcher melting temperature (Catcher-Tm) to detect the presence of the target analyte.

High multiplicity in a single channel using Catcher -Tm analysis

3. Catcher-Tm : Flexibility in the adjustment of Tm value

One unique feature of TOCE™ is the "Catcher," which is a fluorescently labeled artificial template and generates the signal for each target sequence. The Catcher-Tm value can be controlled by adjusting the sequence and length of the Catcher. For TOCE™ assay optimization, the Catcher-Tm value can be easily adjusted and is not limited by the target sequence.

Catcher-Tm : Flexibility in the adjustment of Tm value

4. Consistent Catcher-Tm value regardless of target sequence variations

Current Tm-based analysis method has different Tm value with occurrence of changes in nucleotide sequence of the pathogen. However, TOCE™ technology allows for a consistent Catcher-Tm value that is not affected by variations of the target sequence.

Consistent Catcher-Tm value regardless of target sequence variationse

5. As sensitive as singleplex real-time PCR

The detection limits of multiple pathogens using TOCE™ technology are similar to those of probe-based singleplex real-time PCR.

Singleplex real-time PCR과 동등한 민감도* 10-plex real-time PCR : 7 analytes + dual target for CT & NG + IC (Chlamydia trachomatis; Neisseria gonorrhoeae; Trichomonas vaginalis; Mycoplasma hominis; Mycoplasma genitalium; Ureaplasma urealyticum; Ureaplasma parvum) ** Singleplex real-time PCR : 1 analyte (C. trachomatis)

6. Quantitative analysis by cyclic-CMTA

Presence of infection in infected pathogens and quantitative analyzed result can be simultaneously viewed by setting cyclic-CMTA point in the Real-time PCR process. Cyclic-CMTA point can be set at 30, 40, 50 cycle in the real-time PCR process, and quantitative analysis can be performed depending on the melting peak due to amount of infected pathogens as shown in the chart of analysis result.

Quantitative analysis by cyclic-CMTA석Multiple quantitative analysis of HPVs can be determined by cyclic-CMTA.

The difference of appearance of the melting peak at the cyclic-CMTA point indicates different titers of target (low, intermediate, and high copy). In this example, the melting peak of HPV 33 appears at the first cyclic-CMTA point (cycle 30) corresponding to high copy number. HPV 16 appears at the second cyclic-CMTA point (cycle 40) corresponding to intermediate copy. HPV 18 appears at the third cyclic-CMTA point (cycle 50) corresponding to low copy. This measurement allows for quantitative analysis, persistence, and clearance of multiple HPVs, aiding in the diagnosis of viral infection associated with incidences of cervical cancer.

7. High multiple point mutation detection in a single tube

TOCE™ technology allows for mutation detection to discriminate even one base difference accurately, enabling detection of clustered point mutations. In this example, signal is generated ONLY when mutant-specific pitcher binds to mutated target sequence, discriminating mutations from wild-type sequence.

High multiple point mutation detection in a single tube
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