NUCLEIC ACID AMPLIFICATION

TESTING
VIRAL SAFETY IN BLOOD TRANSFUSION

 Risk of transmitting infection to recipients has

been drastically reduced in the past decades, due
to
a)Improved donor selection
b)Sensitive serologic screening assays
c)Application of viral inactivation procedures during
manufacturing of plasma products
RESIDUAL RISK
 Major sources of remaining risk are:
1. Window period donation
2. Viral variants not detected by current assays
3. Immunosilent donor
4. Laboratory testing error
RESIDUAL RISK
 The greatest threat to the safety of blood supply
is the donation by seronegative donors during the
infectious window period
 Window period donation account for 90% or
more of the residual risk (Report of the
Interorganization Task Force on NAT Testing of
Blood Donors, 2000)
WINDOW PERIOD
 Period precedes the development of antibodies
during the initial infection
 Eclipse phase of the window period - the very
initial phase after exposure when virus replication
is restricted to tissue sites and there is no
detectable viraemia
 Infectious phase of window period is after eclipse
and before seroconversion
NUCLEIC ACID AMPLIFICATION TESTING

 Amplifying a single or a few copies of a piece

of DNA across several orders of magnitude,
generating thousands to millions of copies of a
particular DNA sequence.
APPLICATIONS IN TRANSFUSION MEDICINE

INFECTI GENOTYPE/PH
ENOTYPE
ONS ANTIGENS

HLA PARENTA
TESTING GE
NUCLEIC ACID TESTING
 Hybridisation based methods
 Probes with markers
 Solid phase
 Liquid phase
 Enzymatic
 Disadvantage
 Limited sensitivity
 Abundant quantity
 Inadequate for donor screening
 Less amenable for automation
 Good for research,not diagnosis
 Amplification Based methods
 Small quantity
 More sensitive
 PCR
 Real Time PCR
 TMA
 NASBA
 MICROARRAY
 SNP
PCR-POLYMERASE CHAIN REACTION
 1983-Kary Mullis(Nobel Prize in 1993)
 Thermal cycling
 Amplify a specific region of a DNA strand (the DNA
target) typically ~10 kilo base pairs (kb)
 Cycles of repeated heating and cooling of the reaction
 DNA melting 
 Enzymatic  replication of the DNA
 DNA generated is itself used as a template for replication
 Chain reaction  -DNA template is exponentially amplified
COMPONENTS
 1. DNA template that contains the DNA region
(target) to be amplified
 2. Two primers 
  complementary to the 3' (three prime) ends of each of the sense
and anti-sense  strand of the DNA target.
 3.DNA polymerase (Taq polymerase from
thermaus aquaticus or another) with a
temperature optimum at around 70 °C.
 Discovering a thermostable enzyme was crucial
 4. Deoxynucleotide triphosphates (dNTPs), the building
blocks from which the DNA polymerase synthesizes a new
DNA strand
 5. Buffer solution, providing a suitable chemical
environment for optimum activity and stability of the DNA
polymerase
 6.Divalent cations, magnesium or manganese ions;
 Generally Mg2+ is used
 Mn2+ used for PCR-mediated DNA mutagenesis,
 Higher Mn2+ concentration increases the error rate during DNA synthesis
 7. Monovalent cation potassium ions
DNA ISOLATION
 Cell disruption or cell lysis
 Removing membrane lipids by adding a detergent.
 Removing proteins by adding a protease (optional but
almost always done).
 Precipitating the DNA with an alcohol — usually ice-
cold ethanol or isopropanol.
 Since DNA is insoluble in these alcohols, it will
aggregate together, giving a pellet upon
centrifugation.
 PCR-STEPS
 Reaction volume of 10–200 μl
 Eppendorf tubes-Small reaction tubes (0.2–0.5 ml volumes)
 Thermal cycler- heats and cools the reaction tubes to achieve
the temperatures required at each step of the reaction 
  Peltier effect - Heating and cooling of the block holding the
PCR tubes by reversing the electric current
 Heated lids to prevent condensation at the top of the reaction
tube
 Older instruments needed layering.
PROCEDURE

.
 PCR consists of a series of 20-40 repeated
temperature changes, called cycles
 each cycle commonly consists of 3 discrete
temperature steps
 The cycling is often preceded by a single temperature
step (called hold) at a high temperature (>90°C),
 One hold at the end for final product extension or
brief storage.
 Initialization step: heating the reaction to a temperature of 94–96 °C, which
is held for 1–9 minutes. (hot-start PCR)
 Denaturation step: This step is the first regular cycling event and consists of
heating the reaction to 94–98 °C for 20–30 seconds. It causesDNA melting of the DNA
template by disrupting the hydrogen bonds between complementary bases, yielding single-
stranded DNA molecules.
 Annealing step:
 The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of
the primers to the single-stranded DNA template.
 3-5 degrees Celsius below the Tm of the primers used.
 Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely
matches the template sequence.
 The polymerase binds to the primer-template hybrid and begins DNA synthesis.
 Extension/elongation step:
 Depends on the DNA polymerase used; 
 Taq polymerase-optimum activity temperature at 72–
80 °C,
 DNA polymerase synthesizes a new DNA strand
complementary to the DNA template strand (by adding
dNTPs that are complementary to the template in 5' to 3'
direction)
 At each extension step, the amount of DNA target is
doubled,
 Final elongation: This single step is occasionally performed
at a temperature of 70–74 °C for 5–15 minutes after the last
PCR cycle to ensure that any remaining single-stranded DNA
is fully extended.
 Final hold: This step at 4–15 °C for an indefinite time may be
employed for short-term storage of the reaction.
 Agarose gel electrophoresis is employed for size separation
of the PCR products.
 The size(s) of PCR products is determined by comparison with a DNA
ladder (a molecular weight marker)
PCR STAGES

 Exponential amplification:
 At every cycle, the amount of product is doubled (assuming 100%
reaction efficiency).
 The reaction is very sensitive: only minute quantities of DNA need to be
present.
 Levelling off stage:
 The reaction slows as..
 DNA polymerase loses activity
 Consumption of reagents such as dNTPs and primers-limiting
 Plateau:
 No more product accumulates
 Exhaustion of reagents and enzyme.
PCR-OPTIMISATION-CONTAMINATION
 The PCR method is extremely sensitive,
 Requires only a few DNA molecules in a single
reaction for amplification across several orders of
magnitude
 Adequate measures to avoid contamination from
any DNA present in the lab environment
(bacteria, viruses, or human sources)
CONTROL OF CONTAMINATION
 2 work areas
 Preparation and handling of pre-PCR reagents and the setup of the PCR reaction
 post- PCR processing, such as gel electrophoresis or PCR product purification
 Pipettes with filter tips
 Fresh  laboratory gloves,
 A  laminar flow cabinet with UV lamp as a work station
 A negative control PCR reaction.
 dUTP and Uracil DNA Glycosylase
 Control reaction is set up in the same way as the experimental
PCRs, but without template DNA added, and is performed
alongside the experimental PCRs.
 Inhibitors
 Heparin
 Hemoglobin
 Lactoferrin
 Protocols should be followed
PRIMER DEFECTS
 Cross annealing to unintended targets
 Primer dimer
 Hairpin
HAIR PINS
 Secondary structures in the DNA
 Result in folding or knotting of DNA template or
primers- decreased product yield or failure of the
reaction.
 Correction
 Primer design that includes a check for potential secondary
structures in the primers
 Addition of DMSO or glycerol to the PCR to minimize
secondary structures in the DNA template
POLYMERASE ERRORS

 Taq has no error-proof-reading activity

 Excision of any newly misincorporated nucleotide base from the nascent
DNA strand that does not match with its opposite base in the
complementary DNA strand.
 High error rate (mutations per nucleotide per cycle) of
approximately 1 in 10,000 bases, which affects the fidelity
of the PCR,
 More if errors occur early in the PCR with low amounts of
starting material,
 Accumulation of a large proportion of amplified DNA with
incorrect sequence in the final product.[2]
 Several "high-fidelity" DNA polymerases, having
engineered 3' to 5' exonuclease activity, have
become available.
 KOD DNA polymerase, a recombinant form
of Thermococcus kodakaraensis 
 KOD1 extracted from Thermococcus litoralis; 
 Pfu DNA polymerase, which is extracted from Pyrococcus
furiosus;
 Pwo, which is extracted from Pyrococcus woesii.
MAGNESIUM CONCENTRATION

 Primers which bind to incorrect template sites are stabilized in the presence of
excessive magnesium concentrations
 Stabilize double stranded DNA and prevent complete denaturation of the
DNA during PCR reducing the product yield.[3][4] 
 Inadequate thawing of MgCl2 may result in the formation of concentration
gradients within the magnesium chloride solution
 Reduce the amount of free magnesium present hence reducing the activity of
the enzyme
 Inc .template concentration,
 dNTPs and the
 presence of chelating agents (EDTA) or
 proteins .[
VARIANTS
 Multiplex-PCR uses several pairs of primers
annealing to different target sequences
 Variable Number of Tandem Repeats (VNTR)
PCR targets areas of the genome that exhibit 
length variation
 Asymmetric PCR
 Nested PCR
 Hot-start PCR
 Helicase-dependent amplification
 Nicking Enzyme Amplification Reaction
 Inverse PCR
 Thermal Asymmetric InterLaced
PCR (or TAIL-PCR)
RT PCR
 For amplification of RNA
 Reverse transcriptase enzyme used to generate c
DNA from RNA
 Then PCR done
TMA-TRANSCRIPTION MEDIATED AMPLIFICATION

 HIV,HCV,WNV
 TARGET OF AMPLIFICATION RNA
 RT AND DNA POLYMERASE IN SAME
REACTION
NUCLEIC ACID SEQUENCE BASED AMPLIFICATION

 Single mixture ISOTHERMAL 410 –NO DENATURING

 RNA template is given to the reaction mixture, the first primer
attaches to its complementary site at the 3' end of the template
 Reverse transcriptase synthesizes the opposite, complementary
DNAstrand
 RNAse H destroys the RNA template (RNAse H only destroys
RNA in RNA-DNA hybrids, but not single-stranded RNA)
 The second primer attaches to the 5' end of the DNA strand
 T7 RNA polymerase produces a complementary RNA strand
which can be used again in step 1, so this reaction is cyclic.
 STRAND DISPLACEMENT AMPLIFICATION
 LIGASE CHAIN REACTION
 PROBE
 HYBRID CAPTURE
 CLEAVASE INVADER ASSAY
REAL TIME PCR
 FLOURESCENT-QUENCHER PROBE
DEGRADATION
 FLOURESCENT QUENCHER PROBE
HAIRPIN UNFOLD
 FLOURESCENT QUENCHER WORKING IN
PROXIMITY
 CYBER GREEN DYE AND MELTING CURVE
 FLOURESCENCE PLOTTED-REAL TIME
ANALYSIS POSSIBLE
MICRO ARRAYS
 MULTIPLE PROBES IN GENE CHIPS
 COMPOSITION OF SPECIMEN
SNP-SINGLE NUCLEOTIDE POLYMORPHISM

 Not needed for infectious disease screening

 Red cell antigen detection
 RFLP
IMPLICATIONS IN BLOOD BANKING
 Work flow design
 Space
 Sample integrity
 Technical expertise
 Storage
 Control of contamination
 Cost
FIRST STEP
 1998
 Transmission of HCV by an intravenous
immunoglobulin preparation
 NAT for hepatitis C virus (HCV) RNA in plasma
pools recommended by the Committee for
Proprietary Medicinal Products (CPMP)
 Accepted by the European Agency for the
Evaluation of Medicine (EMEA)
HCV
 Prolonged high-titre viraemic phase before
seroconversion and elevation of ALT, 7-12 weeks
after infection
 Very short doubling time of 2-3 hours, therefore
high viral load titres are achieved
HCV
 Very amenable to detection by pooled NAT
 NAT theoretically reduce the window period by
41-60 days
HCV
HIV
 Short doubling time of 21 hours
 Window period of 16 days (p24 antigen) may be
reduced to 11 days by NAT
HIV
HBV
 HBsAg become positive 50-60 days after
infection
 Preceded by a prolonged phase (up to 40 days) of
low-level viraemia
 Long doubling time of 4 days
 NAT pooling will only detect a small proportion
of this pre-HBsAg window period
HBV
 HBsAg become positive 50-60 days after
infection
 Preceded by a prolonged phase (up to 40 days) of
low-level viraemia
 Long doubling time of 4 days
 NAT pooling will only detect a small proportion
of this pre-HBsAg window period
HBV
INTERNATIONAL FORUM ON IMPLEMENTATION OF DONOR SCREENING
FOR INFECTIOUS AGENTS TRANSMITTED BY BLOOD BY NAT

 Vox Sang 2002;82:87-111

 Countries screening HBV DNA: Japan, Germany
(some plasma manufacturers)
 Countries screening HCV RNA: Australia, New
Zealand, Japan, USA, Canada, Germany, France,
Austria, Italy, Netherlands, UK, Finland, Norway,
Spain(partial), HK
INTERNATIONAL FORUM ON IMPLEMENTATION OF DONOR SCREENING
FOR INFECTIOUS AGENTS TRANSMITTED BY BLOOD BY NAT

 Countries screening HIV RNA: Australia, New

Zealand, Japan, USA, Canada, France,
Netherlands, Spain (partial), Germany (plasma
products only), HK
 Still considering: Sweden, Brazil, Greece, South
Africa
MINI POOL NAT
THANK YOU