New Diagnostics Pipeline. David L. Dolinger, Ph.D.

Transcription

1 New Diagnostics Pipeline David L. Dolinger, Ph.D.

2 Questions What do we need to know to have actionable information from a diagnostic for detection of drug resistance? Specific mutational information Mutational linkage What is the purpose of the solution? Detection of validated resistance mutations Detection of all known resistance mutations Where is the solution needed? Laboratory infrastructure requirements How close to the patient does the testing have to be? Is it dependent upon the test Is it dependent upon the ability to provide drugs What is the critical turn around time? How does this tie to the closeness to the patient How much time will a patient spend waiting What are the throughput requirements? How does this tie to the closeness to the patient What level of detection is required Mixed infections What LoD is significant

3 Concerns for Thought Sample matrix Decontamination Stability Transportation Quantity Extraction Matrix Quality and yield of extraction Combined with Amplification and Detection Amplification Thermal cycling Isothermal Combined with Detection Detection Optical Fluorescence Luminescence Visual Enzymatic Electrochemical

4 Hardware Solutions for Resistance Testing in MTB A Sampling Life Technologies QuantumDx Oxford Nanopore Veredus Life Technologies Great Basin Spartan Biosciences Ahram biomérieux/biofire Micronics Epistem Siemens Roche/IQuum NEC Cepheid Life Technologies Enigma Diagnostics GenMark Dx Life Technologies Integrated Nano- Technologies Atlas Genetics Molbio

5 Chemistry Solutions for Resistance Testing A Molecular Sampling Molecular Amplification (Cyclic or Isothermal) plus Detection Methodology Real time based TaqMan FRET Probes High Resolution Melt Curve Molecular Beacons Standard, Sloppy.... PANA S-Melting Analysis Tagging Oligonucleotide Cleavage Extension Lights On/Lights Off Hybridization based Array Line Probe Sequencing Sanger Next Generation Hybrid Phage & Molecular Pyrazinamidase Physiochemical Analysis HPLC CE Mismatch Analysis by HPLC and melt Heteroduplex analysis

6 Next Generation Sequencing Process DNA Sample NGS Instrument Data Library Preparation Sequencing Data Analysis

7 Where Does It Begin? Sample Matrix Sputum How s Render non-hazardous Transport Deal with it as an inconsistent, heterogenous sample One potential solution Obtain high quality nucleic acids (RNA/DNA) from clinical or environmental specimens Stabilize and preserve 'naked' RNA/DNA for prolonged time periods Inactivate infectious biological pathogens for safe handling and transport Compatible with commercially available RNA/DNA isolation kits Allows storage or transport of specimens either frozen, refrigerated or at ambient temperatures

8 Standardized Sample Prep and Library Creation One possible solution Battery powered GPS, wireless and WiFi Fully automated sample to result platform Low cost cassette drive with the capability of building in library creation Yields high quality DNA that is sequencing ready Integrated Nano Technology

9 How Close Are We? How close do we need to be? What gaps exist in our understanding of the relationship between genotypic and phenotypic information? We have solutions today for centralized laboratory detection of resistance related mutations

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