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Transcript of Nucleic Acid Testing
NUCLEIC ACID AMPLIFICATION TESTING
VIRAL SAFETY IN BLOOD TRANSFUSION
Risk of transmitting infection to recipients has been drastically reduced in the past decades, due toa)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
INFECTIONS
GENOTYPE/PHENOTYPE ANTIGENS
HLA TESTING
PARENTAGE
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
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