Molecular Diagnostics

T. Mazzulli, MD, FRCPCDepartment of MicrobiologyUHN/Mount Sinai Hospital

October 19th, 2009

Objectives

Briefly 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 Backbone

A ribose or a deoxyribose A negatively charged phosphate groupDNA has an H at C2 so become “deoxy”RNA has a hydroxyl at C2C1 linked to a purine or pyrimidine baseCounter-parallel strands bind by H-bonding between nucleotides

http://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/basepair.html

The Pyrimidines      The Purines

Components of DNA/RNA

Base Nucleoside:Base + Ribose

Nucleotide:Base + Ribose + Phosphate

Cytosine Cytidine (C) Cytidine monophosphate (CMP)

Uracil Uridine (U) Uridine monophosphate (UMP)

Thymine Thymidine (T) Thymidine monophosphate (TMP)

Adenine Adenosine (A) Adenosine monophosphate (AMP)

Guanine Guanosine (G) Guanosine monophosphate (GMP)

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

What’s the point of all this? Genetic information is grouped into codons

Codons 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/Annealing

Sequences of DNA that anneal to target DNA or RNA– Must be complimentary– Primers and probes are therefore

specific for the targetNo nucleic acid amplification

tatgcattatttaaaggg5’ 3’

3’----atacgtaataaatttccc----5’

Target

Probe

Molecular Methods

Critical to all molecular tests is extraction of RNA/DNAKey steps include extraction, amplification, detectionMay be used for:– Diagnosis– Monitoring– Screening

Three Methods:– Target amplification– Signal amplification– Probe amplification

Target (Nucleic Acid Amplification Tests) Amplification Methods

1. Polymerase Chain Reaction (PCR):– PCR– Reverse transcriptase (RT)-PCR– Real time (RT)-PCR– Nested PCR– Multiplex PCR– Qualitative/Quantitative PCR

2. Transcription-mediated amplification methods (TMA, NASBA)

3. Strand displacement amplification (SDA)

Polymerase Chain Reaction

Four key steps:1. Denature (Melt) DNA2. Anneal primers (20-25 nucleotides) to DNA target3. Extension of DNA using target DNA template4. 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 efficientRequires excess of DNA polymerase, equimolar dNTPs, deoxyribonuleotide triphosphates (dNTPs), MgCl2 and buffer

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

PCR: Drawbacks False positives Chance of contamination Need equipment and training May 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

RNADNA primers

Reverse transcriptase

ssDNA

PCR step

RNA with Secondary structureMelt

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

Round I

PCR15-30 cycles

Amplicon 1

Primerset 1

Round II Primer set 2

PCR 20-30 cycles

Amplicon 2

Real Time PCR: SYBR Green

Real Time PCR: TaqMan probes

Real Time PCR

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

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 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 Amplicon

Open 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 amplicon

Agarose gelelectrophoresis

Stain with EtBRImage on UV lightbox

Probe or DNA-bindingchemical Automated imaging

system

Denaturelabeled amplicons

Hybridize with captureProbe in 96 well plate

Detect 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 Assays

Viral:– HCV RNA, HBV DNA, HIV-1 RNA, CMV DNA, HPV

DNA, WNV RNABacterial:

– Chlamydia trachomatis/Neisseria gonorrheae, Mycobacterium tuberculosis

Fungal:– None

Parasites:– None

Transcription Mediated Amplification (TMA)

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)

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

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 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

Commercially Available SDA-based Assays

Viral:– CMV DNA

Bacterial:– Chlamydia trachomatis/Neisseria

gonorrheaeFungal:

– NoneParasitic:

– None

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

Cycle Threshold for Detection of DNA

Measuring HBV DNA

Gish and Locarnini, Clin Gastro Hep 2006

Nucleic Acid Amplification Tests (NAAT) for Detection of RNA/DNA

Quantitation 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 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%

Signal Amplification Methods

Branched 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 labelled probes by incubating with dioxetane which emits light in presence of APRemove non-specific hybridization isoC and isoG in preamplifier and label probes which recognize each other but not native C and G

Commercially Available bDNA Assays

Viral:– HBV DNA, HCV RNA, HIV-1 RNA

Bacterial:

Hybrid Capture Assay

Multiple AP conjugates bind to each hybrid molecule and amply the signalDetect complex with chemiluminescent substrateIntensity of emitted light proportional to amount of DNA in reaction

ds DNAdenature

ss DNAhybridize withRNA probe

Capture hybrids

APAP

Commercially Available Hybridization Assays

Viral:– HBV DNA, HPV DNA

Bacterial:– Chlamydia trachomatis/Neisseria gonorrheae

Probe Amplification Assays

Ligase Chain Reaction (LCR)Cleavase-Invader TechnologyCycling probe technology

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 amplificationAt a point mutation the oligos only completely anneal to the mutant sequences and DNA ligase will not ligate the two oligos of each pair togetherWithdrawn from Chlamydia trachomatis testing

Cleavase Invader Technology

Produced by Third Wave Technologies-bought by PEBIO Invader displaces 5’ of bound probeFEN-1 family of themostable DNA polymerases cleaves 5’ overlapCan 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

Target sequence3’ 5’5’

Invader oligo

5’

3’

Probe oligo

Cleavase

5’ 3’3’ 5’

5’

Cleaved probe

Limitations of Molecular Assays

DNA/RNA extraction is criticalSpecimen type: Plasma or serum may be acceptable; tissue, fluids, etc. may notPlasma/serum must be separated and either frozen or tested within 4 to 6 hoursExpensive equipment is usually required

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.)

Genotyping Assays

Pros Cons

Sequencing Discovers Labor-intensivenew mutations Low sensitivity

(15-20% pop.)

Line Probe High throughput Detects knownHigh sensitivity mutations only(5-10% pop.)

Marker lineConj.cont.Amp.cont.

L180

M180

M204

V204

I204

V207L207

M207

I207

Amplified Amplified targettarget

StreptavidinStreptavidin

BiotinBiotin

Alkaline Alkaline PhosphatasePhosphatase

Purple Purple precipitateprecipitate

ChromogenChromogen(NBT/BCIP)(NBT/BCIP)

DNA-probeDNA-probeNitrocellulose Nitrocellulose

stripstrip

InnoLiPA Principle

InnoLIPA HBV Drug ResistanceM arke r line

C onj. C on tro lA m p.C ontro lL8 0 W TV 80 M u tant80 M u tant

V 173 W T G 173 W TL1 73 M utan tL1 80 W TM 180 M u ta ntA 181 W TT18 1 M utan tV 181 M utan tM 204 W TV 204 M utan t204 M utan t

S 204 M utan tN 236 W TT23 6 M utan t

I

I

1 - 2 - 3 -4 - 5 - 6 - 7 - 8 - 9 -

10 - 11 - 12 - 13 - 14 - 15 - 16 - 17 - 18 - 19 -

Molecular Diagnostics: Summary

NAAT is critical to patient managementOf the many NAAT tests available, PCR, bDNA and TMA remain most popular– Sensitivity and dynamic range varies between assays– Standardization allows (to some degree)

interchangeability of the results with different assays

Resistance/Genotyping requires amplification first– Increasing role in making treatment decisions as more

drugs become available

Thank you for your attention!