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    Add as FriendAN INTRODUCTION OF NUCLEIC ACID ASSAYS

    by: NALASIVA NANDA

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    1 : AN INTRODUCTION TO NUCLEIC ACID AMPLIFICATION (NAT) ASSAYS N.Nanda Gopal Jr.Scientist
    2 : What are the situations make confuse? Ø RIBA intermediate reactants. Ø False negative due to - Window period donations. - Chronic carrier state in asymptomatic donor test negative on antibody screening. - Atypical genetic variants of the viruses due to frequent changing of antigenicity. - Immunosuppressive patients may leads to false-negative results. - Laboratory errors.
    3 : Make it clear to ensure blood safety! Ø Implementation of new tests to narrow down the window period. Ø Combining more than 1 or 2 assays complement each other and decrease false positives (FP) and false negative (FN) results. Ø To bring the low incidence for transfusion transmitted infections.
    4 :   We need a Gold standard technique! Ø To eliminate the risks / false negative and to achieve near “ZERO” risk. Ø Nucleic acid amplification technique (NAT) i.e. by Polymerase Chain Reaction (PCR). Ø Licensed by Food and Drug Administration (FDA) for blood and blood products. Ø To confirm the presence of antigen in low titer.
    5 : Over view of NAT Collection of blood sample Separation of blood component Isolation of antigen Subjected to Thermal cycler Amplify the target sequences Analysis.
    6 : What is Polymerase Chain Reaction (PCR)? Ø An Invitro exponential amplification of target nucleic acids (DNA / RNA). Ø Mediated by thermo stable enzyme using primers, flanked either side of the target. Ø Amplified product is called “Amplicon”. Ø Versatile technique with high speed, efficiency, sensitivity and specificity. Ø Eliminates the laborious conventional culture methods.
    7 : Development of PCR Invented by Kary B. Mullis (1985), Cetus corp, USA. Used klenow fragment of DNA polymerase-I, sensitive to high temps > 900C. Ø Replaced by Lawyer et.al. (1989) with Taq DNA Polymerase of Thermus aquaticus, a volcanic achaebacteria.
    8 : Contd.. Ø Recently thermostable, high fidelity with proof reading function were identified.   ·      Vent polymerase- Thermococcus litoralis (Avalable from New England Labs, Baverly). ·      Pfu polymerase- Pyrococcus furiosus (Available from Stratagene, Lajolle). ·      Amplification of nucleic acid over 1 million times so that we can detect by conventional means (Eg. Gel electrophoresis).
    9 : ·      Hot-start technology: o   Taq enzyme is modified to inactivate at ambient temperature. o       Prevents extension of non-specific sequences. o       Primer-dimer formation at initial temperatures. o       Activated by 15 min incubation at 950C, then the PCR protocol starts.  
    10 : NAT background Ø 1985: PCR is used in qualitative manner. Ø Early 90’s: PCR in molecular diagnostics to detect contamination of blood and blood products. Ø Mid 90’s: Wide spread use of different EIA kits leads to results which are difficult to interpret. Ø Last 90’s: Programme of standardization and mandatory testing of PCR in developed countries like Japan, Canada.
    11 : How much sensitive the EIA assays? HCV seroconversion after blood transfusion. Ø 1 st generation ELISA – 2 to 16 weeks – sensitivity 70% to 80%. Ø 2 nd generation ELISA – 10 weeks - sensitivity 92% to 95%. 3 rd generation ELISA – 7 to 8 weeks - sensitivity 97%. By PCR - approx 3 weeks * Sensitivity based on the findings and detection of HCV RNA by PCR.
    12 : Reduction in window period by Introduction of NAT!
    13 : Conventional Reverse Transcription PCR (RT-PCR) Ø Reverse transcription to make the RNA into cDNA. Ø Amplification of cDNA target sequences by PCR using specific primer pairs. Ø Needs post-PCR processing for analysis i.e. by gel electrophoresis. Ø Only qualitative analysis method.
    14 : Disadvantages: Ø End point detection of results. Ø Chances of contamination is high, leads to non-specific amplicions. Ø Specificity is comparatively low. Ø Would not reveal about the each sample in each cycle. Ø Exact quantitation of nucleic acids is not possible. Ø Sensitivity is up to 100 copies / ml or equivalent to 50 IU.
    15 : Real time RT-PCR Ø Uses conventional RT followed by PCR with florescent reporter dye and quencher. Ø Specifically hybridize to the amplicon emits the fluorescence. Ø Directly proportional to the target nucleic acid present.
    16 : Advantages: Ø Amplification, detection and quantitation of PCR product occur simultaneously in the same reaction vessel. Ø Monitors the fluorescence emitted during the reaction in each cycle. Ø Allows convenient quantitation. Ø Eliminates post-PCR manipulation, traditional gel electrophoresis. Ø Reduces carry over contamination. Ø Sensitivity reached up to 100 copies / ml. Ø More accurate quantification.
    17 : NAT standardization Ø Assures the quality of data from testing laboratory. Ø Samples calibrated through international standards (WHO or NIBSC). Ø Inter laboratory variation (Both sensitivity and specificity). Ø Intra-laboratory variation (Day to day variation, different operators etc..,). Ø Use of MB grade reagents and plastic ware. Ø Collection of information regarding procedure, methodology from reference laboratories.
    18 : Biological reference materials Standardization, quality control and potency estimation of biological products. Ø Calibrated in units of biological activity established by consensus following extensive internal studies. Use of commonly accepted international standards (WHO or NIBSC). Ø We used reference standards in the following units.  §  HCV RNA transcript 3 x 10 –8 copies per ul. §   HBV plasmid 6 x 10 –11 copies per ul. §  HIV RNA extracted from cell supernatant 1x 10 –8 copies per ul.
    19 : Quality control (QC) and Quality Assurance (QA) of NAT §  Each test should be including known positive, negative and reagent control. § Validation should be done for each new lot of reagents includes probe and Primers. §   Check the integrity of the sample prior to amplification. §   Established external proficiency panels and programme.  
    20 : Proficiency studies §  Test performance of validated assay. §  Ensure reproducibility of assay. § Confirm skill of laboratory personnel performing the assay. §  QC studies. § Participation in External Quality Assessment Systems (EQAS). §   Implementation of national quality systems.    

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