Sensitivity vs Specificity

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1 Viral Detection Animal Inoculation Culturing the Virus Definitive Length of time Serology Detecting antibodies to the infectious agent Detecting Viral Proteins Western Blot ELISA Detecting the Viral Genome RNA viruses DNA viruses

2 Dengue guidelines for diagnosis, treatment, prevention and control. WHO new edition, 2009

3

4 Sensitivity vs Specificity Sensitivity Proportion of individuals with disease that produce positive test result Highly sensitive: few false negatives Specificity Proportion of individuals without disease that give a negative test result Highly specific: few false positives

5 Serology Detecting antibodies to the infectious agent Western Blot & ELISA Detecting Viral Proteins Detecting the Viral Genome (RNA viruses, DNA viruses) Polymerase Chain Reaction

6 Rapid Quantitative Sensitive ELISA enzyme-linked immunosorbent assay Direct Indirect Sandwich

7 Detection Colorimetric Fluorescent Chemiluminescent

8 ELISA (indirect)

9 After R. A. Goldsby, T. J. Kindt, B. A. Osborne, Kuby Immunology, 4th ed. (W. H. Freeman and Company, 2000), p Microplate-Assay-Kit-ab html#Frataxin-Kitsab gif

10 IgM antibodies IgG First antibodies to be produced IgM antibody levels decline with time Rise during infection Generally present throughout the life of the animal

11 Dengue guidelines for diagnosis, treatment, prevention and control. WHO new edition, 2009

12

13 Western Blot Advantage: Specificity Disadvantages:

14 Western Blot

15 eral_wb

16 Western Blot RML:PK IBH ctrl dpi mw pk myoblasts myotubes

17 Nucleic Acid Detection ag fg pg ng ug mg Visible Ppt (EtOH) Visible pellet Visible by EtBr Staining Radioactive Probe PCR (30-40 cycles) PCR (Nested)

18 Is There a Correct Protocol?? Proteinase K Phenol Salt Extraction Subcellular Fractionation Silica Anion Exchange Cesium Chloride Gradients CTAB Guanidinium thiocyanate RNasin

19 Components of a Cell Small Molecules (w/w) 70% water, 3% sugars 2% lipids (monomers) 0.4% amino acids (monomers) 0.4% nucleotides (monomers) Big Molecules 22% (w/w) proteins, nucleic acids, polysaccharides

20 DNA Isolation Tissue Homogenization Shearing Inactivation of Nucleases (DNAses) Removal of Contaminants Protein RNA DNA concentration Ethanol precipitation Binding to matrix 23 kb kb-- Non-denaturing Agarose gel

21 RNA Isolation Tissue Homogenization Inactivation of Nucleases (RNAses) Endogenous Exogenous Removal of Contaminants Protein DNA RNA concentration Ethanol precipitation Binding to matrix Selective precipitation --28S rrna --18S rrna --trna Formamide agarose gel

22 Tissue (fresh) Cells Blood Tissue Culture Bacteria Soft Tissue Liver Brain Hard Tissue Skeletal muscle Plant Buffer Extract Homogenize Freeze/Powder (mortar & pestle) Tissue mincer

23 Polymerase Chain Reaction Denature Primer Polymerase

24 PCR Hybridization Reaction containing Synthetic DNA (oligonucleotide primers) DNA polymerase Thermostable e.g. Taq polymerase

25 Repeat times A PCR Cycle Denaturation 95 o C for 15 seconds Annealing 55 o C for 30 seconds Extension 72 o C for 45 seconds

26 Primers PCR Requires. Some knowledge of the sequence you wish to amplify synthetic oligonucleotides nt dntps MgCl 2 Thermostable polymerase Template DNA Thermocycler

27 Microfuge Tubes Equipment tight sealing tubes thin walls not affected by high temperature Thermocycler automated

28 Length of Amplification Product Shorter More efficient < 1000 bp Relatively easy > 5000 bp More difficult Extension PCR/ Long Extension PCR Mixture of 2 polymerases

29 Number of Cycles Dependent upon target DNA concentration single copy target (mammalian genome): cycles 40 cycle limit! Taq, dntps, primers become limiting Greater sensitivity required? Nested PCR

30 Nomenclature Real Time PCR qpcr RT PCR reverse transcription PCR q RT PCR = real time PCR Specialized thermocycler Fluorescent reporter probe

31

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