Published on August 4, 2014
PowerPoint Presentation: Widal test ? WIDAL TEST: ? WIDAL TEST Agglutination test employed in the serological diagnosis of enteric fever Like Typhoid ( Salmonella typhi ) & Paratyphoid( Salmonella paratyphi ) PowerPoint Presentation: Salmonella typhi Rod shaped, Flagellated , Aerobic, Gram - ve Bacilli. H( flagella ) antigens O (somatic) antigens : H( flagella ) antigens O (somatic) antigens Antigenic structure of Salmonella PowerPoint Presentation: Discovered by Georges Fernand Isidore Widal , a French Physician and Bacteriologist. Performed in tube or Slide. Observation of agglutination. PowerPoint Presentation: O (somatic) antigens H (flagella) antigens LPS in the cell wall; Heat stable Less immunogenic Agglutination with antisera: Fine, compact, granular chalky clumps Present in flagella; Heat labile; Strongly immunogenic ; Induce rapid & High Ab titres ; Agglutination with antisera: Large, loose, cotton wool clumps PowerPoint Presentation: Somatic Ag of S.typhi -- O Flagellar Ag of S.typhi -- H Somatic Ag of S.paratyphi -- A Flagellar Ag of S.paratyphi -- B PowerPoint Presentation: agglutination test. Detects anti O and H or A and B antibodies in serum Diagnosis of Typhoid and Paratyphoid cases PRINCIPLE: PRINCIPLE Somatic Ab (Test serum) + Somatic Ag O / A Flagellar Ag H / B Agglutination (Positive reaction) : Materials required: Materials required 1. Widal test kit 2. Widal test slide 3. Suspected serum sample 4. Pipette 5. Applicator stick PowerPoint Presentation: ‘O’ ‘H’ ‘PC’ ‘A’ ‘B’ ‘NC’ V PowerPoint Presentation: The circles were labeled as “H”, “O”, “A”, “B”, positive control and negative control from the first circle to the last. One drop of undiluted serum was placed in the first four circles with the help of a sterile pasture pipette. PowerPoint Presentation: One drop of postive control serum and one drop of negative control serum was placed in the fifth and the last circle respectively. One drop of H antigen was added to the first circle and one drop of Salmonella typhi O antigen was added in the second circle. Salmonella paratyphi A antigen and Salmonella paratyphi B antigen were added in the third and fourth circles respectively. PowerPoint Presentation: One drop of ‘H’ antigen was placed at the fifth circle and sixth circle. With separate applicator sticks serum and the antigen was mixed together and was spread well to fill the whole of the individual circle. Then slide was observed for agglutination. LIMITATION: LIMITATION I st week negative. Titers raise in 2nd week EARLY DETECTION IS NOT POSSIBLE ? NEED EARLY DETECTION: ? NEED EARLY DETECTION To avoid spreading To start the treatment Extraction of DNA from Blood Samples: Extraction of DNA from Blood Samples One mL of blood containing 20 mM potassium EDTA as anticoagulant was centrifuged at 10,000 rpm for 5 minutes . Plasma was separated for serology. One mL of lysis buffer (0.2% Triton X100 in Tris HCl pH 8.0) was added to the pellet. The mixture was gently aspirated several times to effect hemolysis . The tube was centrifuged at 12,000 rpm for 6 minutes, the supernatant was discarded, and the procedure was repeated once. PowerPoint Presentation: The pellet was washed once with distilled water. After the removal of the supernatant, the pellet was resuspended in 20-30 µL of distilled water. The tubes were sealed, kept in boiling water for 20 minutes, and brought back to room temperature before being used as a template for PCR. PCR (Polymerase Chain Reaction): PCR (Polymerase Chain Reaction) The polymerase chain reaction ( PCR ) is a biochemical technology in molecular biology used to amplify 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 . PowerPoint Presentation: Extracted DNA + Primer Full DNA Sequence PROCEDURE: PROCEDURE A basic PCR set up requires several components and reagents.These components include: DNA template that contains the DNA region (target) to be amplified. Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C. PowerPoint Presentation: Deoxynucleoside triphosphates ( dNTPs , sometimes called " deoxynucleotide triphosphates "; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand. Buffer solution , providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. ADVANTAGES: ADVANTAGES Very high sensitivity and Specificity. control of the disease. DISADVANTAGES: DISADVANTAGES extensive infrastructure and specialized skills, the PCR facility cannot be made available everywhere, especially in developing countries.