Molecular Diagnostics – How To Get Started

1. Molecular Diagnostics – How To Get Started Danny L. Wiedbrauk, Ph.D. Warde Medical Laboratory Ann Arbor, Michigan

2. Molecular Diagnostics • Fastest growing area in laboratory medicine. • Increasing numbers of: • Detection technologies • Commercial detection kits • Analyte specific reagents • Instrumentation options

3. Growth Projections Cook, SC. Advance for Administrators of the Laboratory, 2007;16(8):56

4. Driving Forces for Change • Nucleic acid detection methods have become an integral and necessary component of laboratory medicine. • Newer technologies are making molecular diagnostic procedures available to a wider range of clinical laboratories than ever before.

7. Traditional PCR Methods Detection Reagent Prep End-point PCR Sample Prep Analysis

8. Traditional PCR Methods • Complex master mixes and amplification profiles • Involved handling amplified nucleic acids • Had increased potential for producing false positive results • Procedures took 1-3 days • Many procedures had subjective interpretations

9. Real-Time PCR –The Enabling Technology

10. What’s So Cool About Real-Time PCR? • Decreased turnaround times • Simultaneous amplification, detection, and data analysis • Closed system • No additions made after specimen is added • Contamination control • Numerical vs. visual readouts • Less subjectivity • Able to monitor amplification efficiency

11. What’s So Cool About Real-Time PCR? • More automation • Some to include automated sample preparation • FDA approved methods • Training and technical support • Decreased QA/QC costs

12. What is Real-Time PCR?

15. PCR Amplification Plot Plateau Linear Phase Fluorescence (Rn) Exponential Phase Threshold CT Baseline Cycle Number

16. 2.3 1-108 virus copies 2.1 1.9 1.7 1.5 1.3 1.1 0.9 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 Cycle number Dilution Series Relative fluorescence

17. TaqMan RT-PCR Results For Enterovirus CSF Urine Serum + Control Cycle Number

18. What This Means to You These new technologies allow general laboratories such as Microbiology and Hematology to do nucleic acid testing.

20. How do we get started?

21. Contamination Control

22. Contamination Control • Target and Probe Amplification tests create millions of new replication-competent molecules. • These amplified nucleic acids (amplicons) can contaminate gloves, skin, clothing, and the environment. • Contamination of a specimen with one amplicon is sufficient to produce a false-positive reaction.

23. Contamination Control • DNA is very hardy and is resistant to many traditional decontamination procedures including: • Alcohols and organic solvents • Detergents and disinfectants • Autoclaving • Drying

24. What Works? • 10% bleach (freshly made) • Ultraviolet light • Nucleolytic agents • Some acids

25. Contamination Control • Engineering controls • Procedural controls • Chemical controls within the assay • Surveillance

26. Work flow Reagent Preparation Specimen Preparation Reaction Setup and Amplification Product Analysis Dedicated Hallway EngineeringControls

27. Engineering Controls - Kits Work flow Amplification, Detection, and Product Analysis Reagent Preparation Specimen Preparation Amplification Setup

28. Real-Time PCR Work flow Amplification, Detection, and Product Analysis Reagent Preparation Specimen Preparation Amplification Setup in Biosafety Cabinet

29. Disposables/Procedural Controls • Aerosol resistant tips • Separate lab coats/gloves • Dedicated equipment • Unidirectional workflow • Only one tube open • Low level positive controls

30. Procedural Controls • Dedicated • equipment • Separate lab coats/gloves

31. Surveillance • Wipe testing • Monitoring negative controls • Monitoring prevalence rates

32. Question • How can you do wipe testing in a lab that routinely grows the infectious agent?

33. Wipe Testing • Wipe testing can reinforce the need to disinfect the environment daily. • Can improve infection control in lab

34. What Skills Do You Need? • Ability to multiple things at once • Fastidious work habits • Able to accurately pipette small fluid volumes • High tolerance for change • Excellent communication skills • Outstanding customer service ethic

35. What Will You Be Doing? • Extracting nucleic acids • Some type of amplification technology • Signal or nucleic acid detection • Interpretation of results

36. Extraction Systems • Remove inhibitory and interfering substances without significantly altering the amount or quality of the target nucleic acid

37. Available Chemistries • Liquid extraction and salting out • Gentra Systems PureGene • Orca Research IsoQuick • Nonspecific binding of nucleic acids to a solid phase • DEAE Dextran • Silica

38. Spin Column Technology A B C D Lysis Bind Wash Elute

39. Qiagen Spin Columns • Silica membrane plus chaotropic salts • No organic extraction • No ethanol precipitation • DNA or RNA, Both • Wide range of clinical samples • Mini, midi, and maxi columns

40. QIAcube Spin-Column Processing

41. Qiagen Clontech Amresco Gentra Systems Stratagene Roche Molecular QIAmp, Rneasy Nucleospin Cyclo-Prep Generation Capture Strata Prep High Pure Commercial Spin Columns

42. Magnetic Particle Technology

43. Cortex Biochem Promega Dynal Roche Molecular MegaZorb MagnaSil Dynabeads DNA Direct DNA Isolation Kit Magnetic Particle Kits

44. Magnetic Separators

45. BioMerieux Mini MagExtractor • Semi-automated extractor for fewer specimens • Magnetic silica particles • Recovery of RNA and DNA from different sample types • 50 l eluate • 24 samples in 1h

46. Qiagen BioRobot EZ1 • Fully automated • Pre-programmed protocol cards • Prefilled, sealed reagent cartridges • Small footprint • 1-6 samples in 15-30 min • 200 to 350 l of sample • Variety of samples

47. Qiagen BioRobot EZ1

48. MagNA Pure Compact System • Fully automated • Barcoded prefilled, sealed reagent cartridges • Variable sample types (100 to 1000 l) • 50 to 200 l elution volumes • 1 to 8 isolations per run • Benchtop; small footprint

49. Major Clinical Systems for Real-Time PCR ABI 7300/7500 Roche LightCycler Cepheid SmartCycler Cepheid GeneExpert

50. Are there many FDA-approved molecular diagnostic tests?