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
´ 1. 2. 3. 4.

Major sources of remaining risk are: Window period donation Viral variants not detected by current assays Immunosilent donor 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 PA ENTAGE .

NUCLEIC ACID TESTING Hybridisation based methods ´ Probes with markers ´ ² Solid phase ² Liquid phase ² Enzymatic ´ Disadvantage ² Limited sensitivity ² Abundant quantity ² Inadequate for donor screening ² Less amenable for automation ² Good for research.not diagnosis .

´ Amplification Based methods « Small quantity « More sensitive PCR ¹ Real Time PCR ¹ TMA ¹ NASBA ¹ MICROARRAY ¹ SNP ¹ .

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 ² Enzymatic replication of the DNA ² ´ ´ DNA generated is itself used as a template for replication Chain reaction -DNA template is exponentially amplified .

´ 3. DNA template that contains the DNA region (target) to be amplified ´ 2. Two primers ´ ¹ complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. ² Discovering a thermostable enzyme was crucial .COMPONENTS 1.DNA polymerase (Taq polymerase from thermaus aquaticus or another) with a temperature optimum at around 70 °C.

the building blocks from which the DNA polymerase synthesizes a new DNA strand ´ 5.4. ´ ¹ ¹ ¹ Generally Mg2+ is used Mn2+ used for PCR-mediated DNA mutagenesis. providing a suitable chemical environment for optimum activity and stability of the DNA polymerase ´ 6.Divalent cations. Deoxynucleotide triphosphates (dNTPs). Higher Mn2+ concentration increases the error rate during DNA synthesis ´ 7. magnesium or manganese ions. Monovalent cation potassium ions . Buffer solution.

giving a pellet upon centrifugation. it will aggregate together.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). ´ Since DNA is insoluble in these alcohols. ´ Precipitating the DNA with an alcohol ² usually ice-cold ethanol or isopropanol.

heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction Peltier effect .2±0.5 ml volumes) Thermal cycler.´ ´ ´ ´ ´ ´ ´ PCR-STEPS Reaction volume of 10±200 l Eppendorf tubes-Small reaction tubes (0. .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.

.

Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. 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.´ Initialization ´ step: heating the reaction to a temperature of 94± 96 °C. It causesDNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases. The polymerase binds to the primer-template hybrid and begins DNA synthesis. ´ 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. . yielding single-stranded DNA molecules.

Extension/elongation step: ´ Depends on the DNA polymerase used. the amount of DNA target is doubled. ´ 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. ´ .

² The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker) . Agarose gel electrophoresis is employed for size separation of the PCR products. Final hold: This step at 4±15 °C for an indefinite time may be employed for short-term storage of the reaction.´ ´ ´ 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.

.

.

.

PCR STAGES ´ Exponential amplification: ² At every cycle. ² The reaction is very sensitive: only minute quantities of DNA need to be present. . the amount of product is doubled (assuming 100% reaction efficiency). ´ Levelling off stage: reaction slows as. ² DNA polymerase loses activity ² Consumption of reagents such as dNTPs and primers-limiting ² The ´ Plateau: ² No more product accumulates ² Exhaustion of reagents and enzyme..

viruses. ´ 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.PCR-OPTIMISATION-CONTAMINATION The PCR method is extremely sensitive. or human sources) ´ .

A laminar flow cabinet with UV lamp as a work station A negative control PCR reaction. . but without template DNA added. dUTP and Uracil DNA Glycosylase Control reaction is set up in the same way as the experimental PCRs.PCR processing. and is performed alongside the experimental PCRs. such as gel electrophoresis or PCR product purification ´ ´ ´ ´ ´ ´ Pipettes with filter tips Fresh laboratory gloves.CONTROL OF CONTAMINATION ´ 2 work areas ¹ ¹ Preparation and handling of pre-PCR reagents and the setup of the PCR reaction post.

Inhibitors ´ Heparin ´ Hemoglobin ´ Lactoferrin ´ Protocols should be followed ´ .

PRIMER DEFECTS Cross annealing to unintended targets ´ Primer dimer ´ Hairpin ´ .

decreased product yield or failure of the reaction. ´ Correction ´ design that includes a check for potential secondary structures in the primers ² Addition of DMSO or glycerol to the PCR to minimize secondary structures in the DNA template ² Primer .HAIR PINS Secondary structures in the DNA ´ Result in folding or knotting of DNA template or primers.

[2] ´ . ´ Accumulation of a large proportion of amplified DNA with incorrect sequence in the final product.POLYMERASE ERRORS ´ Taq has no error-proof-reading activity ² 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.

a recombinant form of Thermococcus kodakaraensis ² KOD1 extracted from Thermococcus litoralis. which is extracted from Pyrococcus furiosus. which is extracted from Pyrococcus woesii. having engineered 3' to 5' exonuclease activity. ² KOD DNA polymerase. have become available. ² Pwo.´ Several "high-fidelity" DNA polymerases. ² Pfu DNA polymerase. .

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.template concentration.[ . dNTPs and the presence of chelating agents (EDTA) or proteins .[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 .

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 ´ .

HCV.TMA-TRANSCRIPTION MEDIATED AMPLIFICATION HIV.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. so this reaction is cyclic. 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. complementary DNAstrand RNAse H destroys the RNA template (RNAse H only destroys RNA in RNA-DNA hybrids. the first primer attaches to its complementary site at the 3' end of the template Reverse transcriptase synthesizes the opposite. .

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) ´ .

therefore high viral load titres are achieved ´ . 7-12 weeks after infection ´ Very short doubling time of 2-3 hours.HCV Prolonged high-titre viraemic phase before seroconversion and elevation of ALT.

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 .

Germany. Germany (some plasma manufacturers) ´ Countries screening HCV RNA: Australia. Spain(partial). Norway. France.INTERNATIONAL FORUM ON IMPLEMENTATION OF DONOR SCREENING FOR INFECTIOUS AGENTS TRANSMITTED BY BLOOD BY NAT Vox Sang 2002. Japan. Finland. New Zealand. UK. HK ´ .82:87-111 ´ Countries screening HBV DNA: Japan. Netherlands. Austria. USA. Italy. Canada.

France. USA. HK ´ Still considering: Sweden. New Zealand. Brazil. South Africa ´ . Canada. Spain (partial). Germany (plasma products only). Japan. Greece. Netherlands.INTERNATIONAL FORUM ON IMPLEMENTATION OF DONOR SCREENING FOR INFECTIOUS AGENTS TRANSMITTED BY BLOOD BY NAT Countries screening HIV RNA: Australia.

MINI POOL NAT .

THANK YOU .

Sign up to vote on this title
UsefulNot useful