Molecular Diagnostics

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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. - PowerPoint PPT Presentation

Text of Molecular Diagnostics

  • Molecular DiagnosticsT. Mazzulli, MD, FRCPCDepartment of MicrobiologyUHN/Mount Sinai HospitalOctober 19th, 2009

  • ObjectivesBriefly review the concepts of DNA/RNA, bases, etc.Review the methodologies available for molecular testing and describe some of the advantages and disadvantagesDiscuss the currently available commercial assays that are available

  • The DNA/RNA BackboneA ribose or a deoxyribose A negatively charged phosphate groupDNA has an H at C2 so become deoxyRNA has a hydroxyl at C2C1 linked to a purine or pyrimidine baseCounter-parallel strands bind by H-bonding between nucleotideshttp://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/basepair.html

  • The PyrimidinesThe Purines

  • Components of DNA/RNA

  • Basepairing is complementaryChargaff'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 prokaryoteshttp://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/basepair.html

  • Whats the point of all this? Genetic information is grouped into codonsCodons are triplets of nucleotidesTranscribed into anti-codon mRNATranslated into amino acids which are the building blocks of proteins http://www.accessexcellence.org/RC/VL/GG/genetic.html

  • Hybridization/AnnealingSequences of DNA that anneal to target DNA or RNAMust be complimentaryPrimers and probes are therefore specific for the targetNo nucleic acid amplificationTargetProbe

  • Molecular MethodsCritical to all molecular tests is extraction of RNA/DNAKey steps include extraction, amplification, detectionMay be used for:DiagnosisMonitoringScreeningThree Methods:Target amplificationSignal amplificationProbe amplification

  • Target (Nucleic Acid Amplification Tests) Amplification MethodsPolymerase Chain Reaction (PCR):PCRReverse transcriptase (RT)-PCRReal time (RT)-PCRNested PCRMultiplex PCRQualitative/Quantitative PCRTranscription-mediated amplification methods (TMA, NASBA)Strand displacement amplification (SDA)

  • Polymerase Chain ReactionFour key steps:Denature (Melt) DNAAnneal primers (20-25 nucleotides) to DNA targetExtension of DNA using target DNA templateDetection 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 efficientRequires excess of DNA polymerase, equimolar dNTPs, deoxyribonuleotide triphosphates (dNTPs), MgCl2 and buffer

  • The PCR workhorse: Taq DNA polymerase DNA polymerase from Thermus aquaticus (Taq polymerase) (Kaledin et al., 1980). The active form is a monomer with a molecular mass of 93.9 kDa (Lawyer et al., 1993). 5'3' exonuclease domain at its amino terminus and C-terminal 5'3' polymerase domain. No 3'5' exonuclease proof-reading activity http://www.probes.com/handbook/figures/0700.html

  • PCR: BenefitsCrude extracts and small amounts of DNA may sufficeDetection of the smallest possible quantities of target DNA in clinical samplesCan be automated and utilized in homogenous assay methodsCan 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

  • PCR: DrawbacksFalse positivesChance of contaminationNeed equipment and trainingMay not be validated for all samples and populations

  • Reverse Transcriptase (RT)-PCRReverse transcriptase will transcribe both single-stranded RNA and single-stranded DNA templates with equivalent efficiency RNA or DNA primer is required to initiate synthesisGenerates 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

  • Nested PCR Increases sensitivity due to high # cyclesIncreases specificity due to annealing in 2nd ampliconContamination risk if tube transfer-can overcomeOften need nucleic acid probe confirmation

  • Real Time PCR: SYBR Green

  • Real Time PCR: TaqMan probes

  • Real Time PCR

  • Multiplex PCRTwo or more primer setsDifferent targets in same reaction tubes produce different ampliconsPrimers must have same reaction kinetics and lack complementarityComplicated to designLess sensitive than single primer set PCR ds DNA

  • Specimen Transport and Storage Affect the AssayCollection, transport, and assay setup must be compatible with the assay Different viruses are stable in different blood components for different timesFor 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-termFor CMV viral load testing virus is stable in blood for 5 days at 4oC or -20oC to -70oC

  • Detection and Analysis of the AmpliconOpen systems vs closed systemsGel analysisColorimetric Microtiter Plate (CMP) system Real-time (homogenous/kinetic) PCRAllele-specific hybridizationDirect sequencing of productDNA microarrays

  • How is the amplicon identified? DNA ampliconAgarose gelelectrophoresisStain with EtBRImage on UV lightboxProbe or DNA-bindingchemical Automated imaging systemDenaturelabeled amplicons

    Hybridize with captureProbe in 96 well plateDetect bound producti.e. biotin-streptavidin

  • Type of sample effects amplification yield May need to boil CSF to release nucleic acidsInhibitory substances in urine such as hemoglobin, crystals, beta-human gonadotropin, nitratesHeparin and small volumes of whole blood inhibit Taq polymeraseAcid citrate in vacutainers can inhibit HIV-1 viral load by 15%- volume effect

  • What are the sources of contamination? Contamination of specimens in NA extraction stepContamination with + control materialCarryover contamination of amplified products

  • Commercially Available PCR-based AssaysViral:HCV RNA, HBV DNA, HIV-1 RNA, CMV DNA, HPV DNA, WNV RNABacterial:Chlamydia trachomatis/Neisseria gonorrheae, Mycobacterium tuberculosisFungal:NoneParasites:None

  • Transcription Mediated Amplification (TMA)

  • Transcription Mediated Amplification (TMA)Reaction occurs isothermally at 41C 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 expressionTranscription 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)

  • Commercially Available TMA-based AssaysViral:CMV DNA, HCV RNA, HIV RNABacterial:Chlamydia trachomatis/Neisseria gonorrheae, Mycobacterium tuberculosis and othersFungal:NoneParasitic:None

  • Strand Displacement Amplification (SDA)

  • 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 materialPrimer containing a restriction site anneals to templateAmplification primers then annealed to 5' adjacent sequences (form a nick) and start amplification at a fixed temperatureNewly 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 reactionsIf target is low with high background DNA, non-specific amplification can swamp system and decrease sensitivity Walker, Linn and Nadeau, Nucleic Acid Research, 1995

  • Commercially Available SDA-based AssaysViral:CMV DNABacterial:Chlamydia trachomatis/Neisseria gonorrheaeFungal:NoneParasitic:None

  • Nucleic Acid Amplification Tests (NAAT) for Detection of RNA/DNALower limit of detection (LLD)Based on probe-it analysisSet at amount of target DNA which is detected >95% of the timeIn general, qualitative assays are more sensitive than quantitative assaysLinearity (Dynamic range) of Quantitative assaysRange of DNA (or RNA) for which the amount can be accurately extrapolated from a standard curve using quantitative standards

  • Cycle Threshold for Detection of DNA

  • Measuring HBV DNAGish and Locarnini, Clin Gastro Hep 2006

  • Nucleic Acid Amplification Tests (NAAT) for Detection of RNA/DNAQuantitation of RNA or DNA may be reported as copies/ml or IU/mlConversion factor for copies/ml to IU/ml is not the same for different assays measuring the same target or different targetsHBV DNA: 5.82 copies/IUHCV RNA: PCR - 2.4 copies/IU; bDNA: 5.2 copies/IUCoefficient of variation (COV) may range from 15 to 50%

  • Signal Amplification MethodsBranched chain DNA (bDNA)Hybrid capture assay

  • Branched Chain DNA (bDNA) AssayMultiple target probes capture target nucleic acid on microtiter wellSecond set of target specific probes bind to targetPreamplifier binds to 2nd set of target probes and/or 8 amplifiersThree alkaline phosphatase-labelled probes hybridize to each branch amplifierDetect labelle