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Molecular Diagnostics

Molecular Diagnostics. T. Mazzulli, MD, FRCPC Department of Microbiology UHN/Mount Sinai Hospital October 19 th , 2009. Objectives. Briefly review the concepts of DNA/RNA, bases, etc.

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Molecular Diagnostics

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  1. Molecular Diagnostics T. Mazzulli, MD, FRCPC Department of Microbiology UHN/Mount Sinai Hospital October 19th, 2009

  2. Objectives • Briefly review the concepts of DNA/RNA, bases, etc. • Review the methodologies available for molecular testing and describe some of the advantages and disadvantages • Discuss the currently available commercial assays that are available

  3. The DNA/RNA Backbone • A ribose or a deoxyribose • A negatively charged phosphate group • DNA has an H at C2 so become “deoxy” • RNA has a hydroxyl at C2 • C1 linked to a purine or pyrimidine base • Counter-parallel strands bind by H-bonding between nucleotides http://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/basepair.html

  4. The Purines The Pyrimidines      

  5. Components of DNA/RNA

  6. Basepairing is complementary • Chargaff's rule explains the amount of adenine (A) in the DNA of an organism, is the same as the amount of thymine (T) and the amount of guanine (G) is the same as the amount of cytosine (C) • The C+G:A+T ratio varies from organism, especially among the prokaryotes http://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/basepair.html

  7. What’s the point of all this? Genetic information is grouped into codons • Codons are triplets of nucleotides • Transcribed into anti-codon mRNA • Translated into amino acids which are the building blocks of proteins http://www.accessexcellence.org/RC/VL/GG/genetic.html

  8. 3’----atacgtaataaatttccc----5’ tatgcattatttaaaggg 5’ 3’ Hybridization/Annealing Probe • Sequences of DNA that anneal to target DNA or RNA • Must be complimentary • Primers and probes are therefore specific for the target • No nucleic acid amplification Target

  9. Molecular Methods • Critical to all molecular tests is extraction of RNA/DNA • Key steps include extraction, amplification, detection • May be used for: • Diagnosis • Monitoring • Screening • Three Methods: • Target amplification • Signal amplification • Probe amplification

  10. Target (Nucleic Acid Amplification Tests) Amplification Methods • Polymerase Chain Reaction (PCR): • PCR • Reverse transcriptase (RT)-PCR • Real time (RT)-PCR • Nested PCR • Multiplex PCR • Qualitative/Quantitative PCR • Transcription-mediated amplification methods (TMA, NASBA) • Strand displacement amplification (SDA)

  11. Polymerase Chain Reaction • Four key steps: • Denature (Melt) DNA • Anneal primers (20-25 nucleotides) to DNA target • Extension of DNA using target DNA template • Detection of amplified product (amplicon) • After n cycles target is amplified by a factor of 2n (e.g. 35 cycles, 235 = 34 billion copies) • Need 30-40 cycles to be efficient • Requires excess of DNA polymerase, equimolar dNTPs, deoxyribonuleotide triphosphates (dNTPs), MgCl2 and buffer

  12. PCR: Benefits • Crude extracts and small amounts of DNA may suffice • Detection of the smallest possible quantities of target DNA in clinical samples • Can be automated and utilized in homogenous assay methods • Can utilize in quantitative and high-throughput assays • Valuable for identifying cultured and non-cultivatable organisms • Used in epidemiology: repetitive elements PCR spacer typing, selective amplification of genome restriction fragments, multilocus allelic sequence-based

  13. PCR: Drawbacks • False positives • Chance of contamination • Need equipment and training • May not be validated for all samples and populations

  14. RNA with Secondary structure Melt DNA primers RNA Reverse transcriptase ssDNA PCR step Reverse Transcriptase (RT)-PCR • Reverse transcriptase will transcribe both single-stranded RNA and single-stranded DNA templates with equivalent efficiency • RNA or DNA primer is required to initiate synthesis • Generates DNA copies of RNAs prior to amplifying that DNA by polymerase chain reaction (PCR) • RNase H activity: RNase H is a ribonuclease that degrades the RNA from RNA-DNA hybrids and functions as an endonuclease and exonuclease • Problems with nonspecific primer annealing and inefficient primer extension due to secondary structure can be overcome by using Thermus thermophilus reverse transcriptase • Kits available for HCV and HIV-1

  15. Round I Primer set 1 PCR 15-30 cycles Amplicon 1 Round II Primer set 2 PCR 20-30 cycles Amplicon 2 Nested PCR • Increases sensitivity due to high # cycles • Increases specificity due to annealing in 2nd amplicon • Contamination risk if tube transfer-can overcome • Often need nucleic acid probe confirmation

  16. Real Time PCR: SYBR Green

  17. Real Time PCR: TaqMan probes

  18. Real Time PCR

  19. Multiplex PCR • Two or more primer sets • Different targets in same reaction tubes produce different amplicons • Primers must have same reaction kinetics and lack complementarity • Complicated to design • Less sensitive than single primer set PCR ds DNA

  20. Specimen Transport and Storage Affect the Assay • Collection, transport, and assay setup must be compatible with the assay • Different viruses are stable in different blood components for different times • For HIV-1 viral load, HCV RNA & HBV DNA, plasma must be separated from cells within 6 hrs and plasma can be stored at 4oC for several days or -70oC for long-term • For CMV viral load testing virus is stable in blood for 5 days at 4oC or -20oC to -70oC

  21. Detection and Analysis of the Amplicon • Open systems vs closed systems • Gel analysis • Colorimetric Microtiter Plate (CMP) system • Real-time (homogenous/kinetic) PCR • Allele-specific hybridization • Direct sequencing of product • DNA microarrays

  22. How is the amplicon identified? DNA amplicon Probe or DNA-binding chemical Automated imaging system Denature labeled amplicons Agarose gel electrophoresis Hybridize with capture Probe in 96 well plate Stain with EtBR Image on UV lightbox Detect bound product i.e. biotin-streptavidin

  23. Type of sample effects amplification yield… • May need to boil CSF to release nucleic acids • Inhibitory substances in urine such as hemoglobin, crystals, beta-human gonadotropin, nitrates • Heparin and small volumes of whole blood inhibit Taq polymerase • Acid citrate in vacutainers can inhibit HIV-1 viral load by 15%- volume effect

  24. What are the sources of contamination? • Contamination of specimens in NA extraction step • Contamination with + control material • Carryover contamination of amplified products

  25. Commercially Available PCR-based Assays Viral: • HCV RNA, HBV DNA, HIV-1 RNA, CMV DNA, HPV DNA, WNV RNA Bacterial: • Chlamydia trachomatis/Neisseria gonorrheae, Mycobacterium tuberculosis Fungal: • None Parasites: • None

  26. Transcription Mediated Amplification (TMA)

  27. Transcription Mediated Amplification (TMA) • Reaction occurs isothermally at 41°C • 109 increase in target RNA in 2 hrs (produces 100 – 1000 copies per cycle) • Since this assay only amplifies RNA it can be used to detect RNA genome and RNA from viable bacteria • Measures replication of DNA viruses by detecting late mRNA expression • Transcription mediated amplification (TMA): RT with own RNAse and T7 polymerase. Detection by hybridization protection assay-2 fluorophors (Gen-Probe) • NA sequence based amplification (NASBA): RT, RNaseH, T7 bacteriophage RNA polymerase. Detect with hybridization with chemiluinescent probes (bioMeriieux)

  28. Commercially Available TMA-based Assays Viral: • CMV DNA, HCV RNA, HIV RNA Bacterial: • Chlamydia trachomatis/Neisseria gonorrheae, Mycobacterium tuberculosis and others Fungal: • None Parasitic: • None

  29. Strand Displacement Amplification (SDA)

  30. Strand Displacement Amplification (SDA) • Isothermal nucleic acid amplification method that relies on two concurrent polymerization steps and the displacement of 1 nicked strand of genetic material • Primer containing a restriction site anneals to template • Amplification primers then annealed to 5' adjacent sequences (form a nick) and start amplification at a fixed temperature • Newly synthesized DNA is nicked by restriction enzyme, polymerase starts amplification again, displacing the newly synthesized strands. • 109 copies of DNA can be made in one reaction • Alleviate non-specific reactions with organic solvents to increase stringency of reactions • If target is low with high background DNA, non-specific amplification can swamp system and decrease sensitivity Walker, Linn and Nadeau, Nucleic Acid Research, 1995

  31. Commercially Available SDA-based Assays Viral: • CMV DNA Bacterial: • Chlamydia trachomatis/Neisseria gonorrheae Fungal: • None Parasitic: • None

  32. Nucleic Acid Amplification Tests (NAAT) for Detection of RNA/DNA • Lower limit of detection (LLD) • Based on probe-it analysis • Set at amount of target DNA which is detected >95% of the time • In general, qualitative assays are more sensitive than quantitative assays • Linearity (Dynamic range) of Quantitative assays • Range of DNA (or RNA) for which the amount can be accurately extrapolated from a standard curve using quantitative standards

  33. Cycle Threshold for Detection of DNA

  34. Measuring HBV DNA Gish and Locarnini, Clin Gastro Hep 2006

  35. Nucleic Acid Amplification Tests (NAAT) for Detection of RNA/DNA • Quantitation of RNA or DNA may be reported as copies/ml or IU/ml • Conversion factor for copies/ml to IU/ml is not the same for different assays measuring the same target or different targets • HBV DNA: 5.82 copies/IU • HCV RNA: PCR - 2.4 copies/IU; bDNA: 5.2 copies/IU • Coefficient of variation (COV) may range from 15 to 50%

  36. Signal Amplification Methods • Branched chain DNA (bDNA) • Hybrid capture assay

  37. Branched Chain DNA (bDNA) Assay • Multiple target probes capture target nucleic acid on microtiter well • Second set of target specific probes bind to target • Preamplifier binds to 2nd set of target probes and/or 8 amplifiers • Three alkaline phosphatase-labelled probes hybridize to each branch amplifier • Detect labelled probes by incubating with dioxetane which emits light in presence of AP • Remove non-specific hybridization isoC and isoG in preamplifier and label probes which recognize each other but not native C and G

  38. Commercially Available bDNA Assays Viral: • HBV DNA, HCV RNA, HIV-1 RNA Bacterial:

  39. ds DNA denature ss DNA hybridize with RNA probe Capture hybrids AP AP Hybrid Capture Assay • Multiple AP conjugates bind to each hybrid molecule and amply the signal • Detect complex with chemiluminescent substrate • Intensity of emitted light proportional to amount of DNA in reaction

  40. Commercially Available Hybridization Assays Viral: • HBV DNA, HPV DNA Bacterial: • Chlamydia trachomatis/Neisseria gonorrheae

  41. Probe Amplification Assays • Ligase Chain Reaction (LCR) • Cleavase-Invader Technology • Cycling probe technology

  42. Ligase Chain Reactions (LCR) • Thermostable DNA ligase to ligate together perfectly adjacent oligos. • Two sets of oligos anneal to one strand of the gene • With a wild-type target sequence, the oligo pairs ligate together and become targets for annealing other oligos in an exponential amplification • At a point mutation the oligos only completely anneal to the mutant sequences and DNA ligase will not ligate the two oligos of each pair together • Withdrawn from Chlamydia trachomatis testing

  43. Cleavase Invader Technology 5’ • Produced by Third Wave Technologies-bought by PEBIO • Invader displaces 5’ of bound probe • FEN-1 family of themostable DNA polymerases cleaves 5’ overlap • Can heat reaction to allow for primer exchange equilibrium ---new uncleaved probes bind • Detect point mutations because can overlap of invader probe can be only 1 bp—track mutations • Generate distinct fragments of different genotypes • Does not increase amount of target sequence- fewer problems of false + and contamination Probe oligo Invader oligo 5’ 3’ 3’ 5’ Target sequence Cleavase 5’ Cleaved probe 5’ 3’ 3’ 5’

  44. Limitations of Molecular Assays • DNA/RNA extraction is critical • Specimen type: Plasma or serum may be acceptable; tissue, fluids, etc. may not • Plasma/serum must be separated and either frozen or tested within 4 to 6 hours • Expensive equipment is usually required

  45. Genotyping • Used for: • Detection of mutations that confer resistance to antiviral agents • Genotyping of isolates for epidemiological purposes; categorizes patient isolates into 8 different HBV genotypes (A to H) and 6 different HCV genotypes (1 to 6 with 24 subtypes) • Methods include: • Sequencing • Hybridization (e.g. Line Probe Assay, Trugene Assay, Invader assay, etc.)

  46. Genotyping Assays ProsCons SequencingDiscovers Labor-intensive new mutations Low sensitivity (15-20% pop.) Line ProbeHigh throughput Detects known High sensitivity mutations only (5-10% pop.)

  47. Conj.cont. Amp.cont. Marker line L180 M180 M204 V204 I204 V207 L207 M207 I207 InnoLiPA Principle Chromogen(NBT/BCIP) Purple precipitate Alkaline Phosphatase Streptavidin Biotin Amplified target DNA-probe Nitrocellulose strip

  48. InnoLIPA HBV Drug Resistance

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