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Frederick L. Kiechle, MD, PhDMedical Director, Clinical Pathology
Memorial Healthcare SystemPathology Consultants of South Broward, LLP
Hollywood, FL 33021
Point-of-Care Molecular Diagnostics: The Future
Outline Molecular Diagnostics 3 steps
Miniaturization for POCT application
Proof of Concept Cepheid GeneXpert Enigma Diagnostics DxNA LLC: GeneSTAT IQUUM: Liat analyzer
DNA Sequencing Genia and Oxford Nanopore
Conclusions
Molecular Diagnostics
AACC CPOCT 9/18/20143
The use of DNA/RNA to test for specific states of disease or health
Infectious DiseasesOncologyPharmacogenomicsGenetic Disease ScreeningPersonalized MedicineCoagulation
Three Basic Steps
4
Extraction & Purification of nucleic acid
Amplification (making copies)
Detection of amplifiedproduct
Real-Time PCR
End-Product Detection
Microarrays
Luminex (similar to flow analysis)
Sequencing
Features of POCT Molecular Device
5
Hand held Portable Cost/test < $25.00 Total time to result < 30 minutes All 3 molecular steps in unit use device Results = clear with no interpretation Accuracy and precision Full process controls (aka internal controls) are extracted, amplified
and detected along with target
Kiechle & Holland. Clin Lab Med 2009; 29:555-560.
Software Features
1. Input user / Patient ID
2. Control assay & System performance
3. Perform data analyses and test interpretation
4. Wireless connectivity
5. Multiplexing and Melting curve analysis
6
Miniaturization Using Microfluidics
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Behavior, precise control and manipulation of fluids constrained to small (submillimeter) scale
Micro features
• Small volume (nL = 10-9 L/ pl = 10-12 L)• Small size• Low energy consumption• Effects of the micro domain
Used in development of
• Inject printheads• DNA chips• Lab-on-a-chip technology
• Sia S, Kricka L. Lab Chip 2008; 8:1982-1983.
POCT Molecular Diagnosis in Canadian Urban Mobile Health Clinics
8
Interviewed 9 mobile clinic directors and supervisors from 6 clinics
All supported further development of rapid molecular testing using: microfluidics microchip-based platform with integrated sample prep, genetic
amplification, and detection on one chip
Device ~ $1,000; disposable microchip $1 each
Infectious disease detection: CT, NG, HPV, HIV
Navid EL, et al. Point of Care 2011; 10:40-44.
POCT: Benefit to Clients
9
Reduced waiting times
Fewer or no follow up visits
Immediate discussion of
results and Rx
Improve healthcare
accessibilityNavid EL et al. Point of Care 2011; 10:40-44
February 2012
Cepheid GeneXpert Features Single step assay
Place premeasured reagents into labeled parts in the cartridge
70 minutes for most assays
Includes sample processing control to monitor reaction steps and inhibitors
Enigma Diagnostics
www.enigmadiagnostics.com
Founded in 2004
Porton Down, Salisbury UK
POC infectious disease CE mark flu A/B + RSV
1/14/14 Development: MDR-TB
February 2012
Assembled Assay Cartridge
ECP Capillary
RSC Special Report. 2009;(No.317):238-244.
DxNA LLC St. George, Utah
POC Infectious Disease
GeneSTAT
+ 2009 A /H1N1 influenza test, not FDA approved, but authorized by FDA for Emergency Use. Used Roche High Pure RNA Isolation Kit
+ Also Avian flu H5N1 test evaluated in Vietnam in 2010
1/2/14 acquired PathoGene LLC: MSSA/MRSA; Flu A/B; Coccidioidomycosis in development
GeneSTATScreen shot and results
Device LoadedCraig Mosman of DxNA in Hanoi 4/25/2010
GeneSTAT Features Portable, battery powered One button operation after sample is loaded Built to be CLIA waved Disposable, pocket-sized, single use plastic unit Patented close system and photometric cell Sonication with high frequency sound waves to lysis cells PCR chemistry is freeze dried; shelf life 6 months at room
temperature CE marked in 2011 Cartridge test ID with RFID embedded
IQUUMLiat™ Analyzer Advantages
Rapid sample-to-result automation with quantitative analysis
Completely closed system eliminates contamination and allows nucleic acid testing in any setting
Flexible platform is adaptable to various assays and analytes
Flexible tube divided into sealed segments
IQUUM Update Purchased by Roche Molecular Diagnostics April 7, 2014
Liat Influenza A/B Assay CE marked and FDA cleared
Cost/Sample:$400K ‐ >$1M $25K‐$50K $1K‐$5K Genia <$100
21
Genia Technologies, Inc.
Mountain View, California
6/2/14 acquired by Roche Diagnostics
Single molecule nanopore-based sequencing by synthesis
Distinguishes 4 bases by detecting 4 different sized tags released from 5´-phosphate modified nucleotides
Sci Rep 2, 682: DOI:10.1038/step00684 (2012).
Genia Technology
Figure 1. Schematic of a single molecule DNA sequencing by a nanopore with phosphate tagged nucleotides. Genia’s NanoTag sequencing approach identifies DNA sequences not by detecting the nucleotides themselves with the nanopore, but by measuring the current changes caused by the passage of each of four different tags that are released from the incorporated nucleotide during the polymerase reaction.
Time (ms)
DNA Sequencing To Go
Oxford Nanopore's MinION device. Credit: Oxford Nanopore
25
Oxford Nanopore
MinION – Disposable DNA Sequencing Size of USB memory stick < $900.00 Currently 2,000 nanopores
2013: 8,000 nanopores – 20 node installation – complete human genome sequence in 15 minutes Specimen – whole blood, no extraction or amplification required
2014: New membrane to support nanopore 10,000 base sequenced at one time
Science 2014;343:829-830
26
Nanopore Sensing
This diagram shows a protein nanopore set in an electrically resistant membrane bilayer. An ionic current is passed through the nanopore by setting a voltage across this membrane. If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current. Measurement of that current makes it possible to identify the molecule in question. For example, this system can be used to distinguish between the four standard DNA bases G, A, T and C, and also modified bases. It can be used to identify target proteins and small molecules, or to gain rich molecular information, for example to distinguish between the enantiomers of ibuprofen or study molecular binding dynamics.
Conclusion
1. The interest in the miniaturization of the three distinct steps in PCR-based molecular methods is currently under intense investigation.
2. Soon, portable, hand-held, inexpensive POCT devices, equivalent to currently offered full-size systems will become available to aid in the detection of mutations or in the identification of infectious agents.
References1. Holland CA, Kiechle FL. Point-of-care molecular
systems – past, present and future. Current Opinion Microbiol 2005; 8:504-509.
2. Kiechle FL, Holland CA. Point-of-care testing and molecular diagnostics: Miniaturization required. Clin Lab Med 2009; 29:555-560.
3. Agrawal N, Ugaz VM. A buoyancy-driven compact thermocycler for rapid PCR. Clin Lab Med 2007; 27:215-223.
The End
FINITO
FINThank You
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