Diagnostic Virology Lecture 2 PDF
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Ibn Sina National College for Medical Studies
Dr. Fatima Salah Mohammed
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Summary
This lecture covers various approaches to diagnosing viral infections, including viral isolation, antigen detection, serology, and molecular methods. It also discusses cell culture techniques and different types of PCR, such as real-time PCR and conventional PCR.
Full Transcript
Dr. Fatima Salah Mohammed Haematopathologist and virologist Important points Viruses are intracellular organisms Very small to be seen directly by light microscope Parvovirus B19 particle size is 22nm the appropriate sample depend on the type of the disease and at what stage Lastly the cost and...
Dr. Fatima Salah Mohammed Haematopathologist and virologist Important points Viruses are intracellular organisms Very small to be seen directly by light microscope Parvovirus B19 particle size is 22nm the appropriate sample depend on the type of the disease and at what stage Lastly the cost and time effective test is the most important point. Approaches to diagnose viruses Viral isolation Viral identification by electron microscope Eg. Ebola virus is dangerous to be cultured Antigen detection Serology (antibody detection) Molecular methods (nucleic acid detection) Cell culture Detect viral growth by the presence of characteristic cell changes called cytopathic effect like increase in cell size (balloning)or change of their shape or cell fusion (herpes viruses causing multi nucleated giant cells) 2. Hemadsorption : attachment of RBCs to virus infected cells (influenza virus) 3. Decrease acid production by infected cells using phenol red(change to yellow in the acidic media) 4. Definitive identification using the following 1. Continue …… Using fluorescent antibodies Hemaglutination inhibition Real time PCR and gene sequencing Antigens and antibodies detection The most common method used is ELISA There is different types : Direct, indirect and sandwich ELISA Nucleic acid detection Either direct hybridization, or after amplification NAATs(nucleic acid amplification tests) NAATs are many types most important of which are: 1-Target amplification 2-Signal amplification Target amplification divided into: 1-Isothermal amplification(NASBA,TMA) 2Thermo cycler amplification(P CR, real time PCR..) Polymerase Chain Reaction Is a technique for DNA or RNA amplification and detection Requirements: 1. DNA template (target sequence) 2. DNA polymerase :must be an enzyme stable at high temp. is derived from Thermus aquaticus 3. DNA primer: is a synthetic DNA strand of about 18-25 nucleotide complementary to 3` of the target DNA (forewod primerand reverse primer) 4. PCR machinei: automatedThermal cycler machine 5. dNTP,mg+2 Steps of PCR Denaturation at 95ْ: separation of double strands,and each strand act as template 2. Annealing : binding of the primers at the 3`of each template this occur at 54 3. Extension :DNA polymerase act at 72ْ to add dNTP at the 3` of the primers This step repeted 30-40 time 1. Types of PCR Conventional PCR: the DNA product detected at the end of the reaction Real time PCR: used to amplify and simultaneously quantify a target DNA ,and is detected as the reaction progress using a fluorescent dye (Taq Man probe) or SYBR green dye Nested PCR: four primers are used ,the product of the first reaction act as template to the next reaction Multiplex PCR: RT- PCR Results are expressed in qualitative manner(detected or not detected) or in quantitative manner( No. of copies or log )
