Porth's Essentials of Pathophysiology, 4e

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Mechanisms of Infectious Disease

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a minimum threshold, the PCR is considered positive and indicates the presence of the target DNA in a specimen. Several variations of molecular gene detection tech- niques in addition to PCR have been developed and incorporated into diagnostic kits for use in the clinical laboratory, including, transcription-mediated amplifica- tion (TMA), strand displacement amplification, hybrid capture assays, and DNA sequencing. Many of the newer gene detection technologies have been adapted for quantitation of the target DNA or RNA in serum or plasma specimens of patients infected with viruses such as HIV and hepatitis C. If the therapy is effective, viral replication is suppressed and the viral load (level of viral genome) in the peripheral blood is reduced. Conversely, if mutations in the viral genome lead to resistant strains or if the antiviral therapy is inef- fective, viral replication continues and the patient’s viral load rises, indicating a need to change the therapeutic approach. Molecular biology has revolutionized medical diag- nostics. Using techniques such as PCR, laboratories now can detect as little as one virus or bacterium in a single specimen, allowing for the diagnosis of infec- tions caused by microorganisms that are impossible or difficult to grow in culture. These methods have increased sensitivity while decreasing the time required to identify the etiologic agent of infectious disease. For example, using standard viral culture, it can take days to weeks to grow a virus and correlate the CPE with the virus. Using molecular biologic techniques, labo- ratories are able to complete the same work in a few hours. DNA Sequencing Originally described in 1976, DNA sequencing has gone through many modifications and has become one of the most powerful tools for laboratory diagno- sis. The most common sequencing method is known as Sanger sequencing. Sanger sequencing uses nucle- otides (similar to PCR) to build a chain of DNA. Terminator dyes that have been fluorescently labeled are inserted into the elongating fragment, causing the PCR reaction to stop at random lengths. The newly labeled double-stranded DNA fragments are broken apart, and separated by size (with a resolution of just one nucleotide) by gel or capillary electrophoresis. The resulting chart indicates the terminator color and the length of the fragment at termination (Fig. 14-12). Sanger sequencing has quickly become the “gold stan- dard” for identification of microbes that cannot be identified by other routine methods. Sequencing has also become the most accurate method for classify- ing microbes into their taxonomic group (Genus and species). While Sanger sequencing has improved diagnosis of infectious diseases, it is still limited to sequenc- ing a very small section of the genome. Consider that Sanger sequencing was used in the first complete sequence of the human genome, which took more

Target DNA

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Heat

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Primers

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Extension of new DNA usingTaq polymerase

Repeat process multiple times

Final amplified products

Detection probe

After many cycles of heating, cooling, and polymeriza- tion, and only if the specific pathogen (or its DNA) is present in the specimen, millions of uniformly sized pathogen DNA fragments are produced. The polymer- ized DNA fragments are separated by electrophoresis and visualized with a dye or identified by hybridiza- tion with a specific probe. A modification of PCR, known as real-time PCR , continues to revolutionize medical diagnostics. Real- time PCR uses the same principles as PCR, but includes a fluorescence-labeled probe that specifi- cally binds a target DNA sequence between the oli- gonucleotide primers. As the DNA is replicated by the DNA polymerase, the level of fluorescence in the reaction is measured. If fluorescence increases beyond triphosphates, new DNA strands are amplified from the point of the primer attachment. The process is repeated many times (called cycles) until millions of copies of DNA are produced, all of which have the same length (defined by the distance [in base pairs] between the primer binding sites). These copies are then detected by electrophoresis and staining or through the use of labeled DNA probes that, similar to the primers, recognize a specific sequence located in the amplified section of DNA. FIGURE 14-11. Polymerase chain reaction. The target DNA is first melted using heat (generally around 94°C) to separate the strands of DNA. Primers that recognize specific sequences in the target DNA are allowed to bind as the reaction cools. Using a unique, thermostable DNA polymerase calledTaq and an abundance of deoxynucleoside