diagnostics and biomarkers

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*Molecular DiagnosticsThe success of modern medicine depends on the detection of specific molecules e.g. Viruses Bacteria Fungi Parasites Proteins In water, plants, soil and humans.


*Characteristics of a Detection SystemA good detection system should have 3 qualities: Sensitivity Specificity Simplicity Sensitivity means that the test must be able to detect very small amounts of target even in the presence of other molecules. Specificity: the test yields a positive result for the target molecule only. Simplicity: the test must be able to run efficiently and inexpensively on a routine basis.


*Immunological Diagnostic ProceduresImmunological diagnostic procedures are often used to: Test drugs Monitor cancers Detect pathogens ELISA (Enzyme Linked Immunosorbent Assay) This involves the reaction of an antibody with an antigen and a detection system to determine if a reaction has occurred. ELISA involves: Binding of the test molecule or organism to a solid support e.g. micro titer plate.


*ELISAAddition of a specific antibody (primary antibody) which will bind to the test molecule if it is present. Washing to remove unbound molecules. Addition of secondary antibody which will bind to the primary antibody. The secondary antibody usually has attached to it an enzyme e.g. alkaline phosphatase. Wash to remove unbound antibody. Addition of a colourless substrate which will react with the secondary antibody to give a colour reaction which indicates a positive result.




*ELISA Animation


*ELISA Lab/Detection of HIV


*Detection of HIV


*DNA Diagnostic SystemsDNA Diagnostic Systems include: DNA Hybridization PCR Restriction endonuclease analysis RAPD (random amplified polymorphic DNA) DNA fingerprinting


*DNA HybridizationBacterial and viral pathogens may be pathogenic because of the presence of specific genes or sets of genes. Genetic diseases often are due to mutations or absence of particular gene or genes. These genes (DNA) can be used as diagnostic tools. This involves using a DNA probe during DNA hybridization. What is a DNA probe? How does DNA hybridization work?


*DNA HybridizationFor DNA hybridization: A probe is needed which will anneal to the target nucleic acid. Attach the target to a solid matrix e.g. membrane. Denaturation of both the probe and target. Add the denatured probe in a solution to the target. If there is sequence homology between the target and the probe, the probe will hybridize or anneal to the target. Detection of the hybridized probe e.g. by autoradiography, chemiluminsence or colorimetric.


*DNA hybridization movie


*Detection of MalariaMalaria is caused by the parasite Plasmodium falciparum. What kind of organism is P. falciparum? The parasite infects and destroys red blood cells. Symptoms include fever, rashes and damage to brain, kidney and other organs. Current treatment involves microscopic observations of blood smears, which is labour intensive. Other methods e.g ELISA does not differentiate between past and present infection. Why?


*Detection of MalariaA DNA diagnostic system would only measure current infection. (Why?) The procedure involves: A genomic library of the parasite was screened with probes for parasitic DNA. The probes which hybridized strongly were tested further. The probes were tested for their ability to hybridize to other Plasmodium species which do not cause malaria and to human DNA.


*Detection of MalariaProbes which hybridized to P. falciparum only could be used as a diagnostic tool. The probe was able to detect 10 pg of purified DNA or 1 ng of DNA in blood smear. Other DNA probes were developed for the following diseases: Salmonella typhi (food poisoning) E. coli (gastroenteritis) Trypanosoma cruzi (chagas’ disease)


*Polymerase Chain ReactionPCR uses 2 sequence specific oligionucleotide primers to amplify the target DNA. The presence of the appropriate amplified size fragment confirms the presence of the target. Specific primers are now available for the detection of many pathogens including bacteria (E. coli, M. tuberculosis), viruses (HIV) and fungi.


*Using PCR to Detect for HIVRT-PCR (reverse transcriptase PCR). HIV has a ssRNA genome. Lyse plasma cells from the potentially infected person to release HIV RNA genome. The RNA is precipitated using isoproponal. Reverse transciptase is used to make a cDNA copy of the RNA of the virus. This cDNA is used as a template to make dsDNA.


*RT-PCR Diagnosis of HIV


*Using PCR to Detect for HIVSpecific primers are used to amplify a 156 bp portion of the HIV gag gene. Using standards the amount of PCR product can be used to determine the viral load. PCR can also be used as a prognostic tool to determine viral load. This method can also be used to determine the effectiveness antiviral therapy. (Brock Biology of Microorganisms 9th ed. pg 883-886).


*DNA Fingerprinting (RFLP)RFLP = Restriction Fragment Length Polymorphism Regular fingerprinting analyses phenotypic traits. DNA fingerprinting analyses genotypic traits. DNA fingerprinting (DNA typing) is used to characterize biological samples e.g. In legal proceedings to identify suspects and clear others. Paternity testing


*DNA Fingerprinting (RFLP)The procedure involves: Collection of sample e.g. hair, blood, semen, and skin. Examination of sample to determine if there is enough DNA for the test. The DNA is digested with restriction enzymes. Digested DNA is separated by agarose gel electrophoresis. DNA is transferred by Southern blotting to a membrane. Membrane is hybridized with 4-5 different probes. Detection of hybridization.

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Last Updated: 8th March 2018

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