Micro Exam 2: Molecular and Rapid Techniques for BacterialID – Flashcards
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| Hypothesis for typing systems? |
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| Series of isolate obtained from an epidemiological cluster are clonally related. |
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| Characteristic of typing systems |
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| It is only useful if there is stability within a strain of organism(identify same organisms) and diversity within a species (differentiate between different organisms within a same species) |
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| Phenotypic systems |
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| based on presence or absence of metabolic/biologic activities. * organsims can alter expression of biologic properties(mutation, environment) so its not as reliable as molecular methods. |
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| Genotypic systems(molecular) uses what as the template? Why? |
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| nucleic acids. Diversity among microbial species. Can show evolutionary divergence. |
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| Advantages of using nucleic acid as a template for ID? |
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| Relatively Stable Ubiquitous in nature (found in all organisms). Purification is identical for all organisms. |
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| PCR - what is it? Advantages? |
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| Target site amplification. Rapidly and exponentially amplify specific DNA sequence. Rapid Sensitive Specific. |
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| Components of PCR |
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| Need a template DNA, 2 primers to replicate DNA (one for each strand, 10-20 bases long), free base pairs, DNA polymerase(TAQ - thermostable, because of the use of heat in the procedure). |
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| Thermocycling |
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| 3 cycles: Heat is up - to denature it, turns into two single strands of DNA. Cool to annealing temperature - binds primers to complementary DNA sequences. Raise temp to 72 - DNA polymerase can start to build new DNA. |
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| Gel Electrophoresis |
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| Separates amplicon - based on size and charge. DNA is negatively charged due to phosphate backbone. Travels to anode (+). |
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| Visualization of Separated DNA - |
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| Add a die that attaches to DNA and fluoresces in UV light. ex. Ethidium Bromide. |
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| Real Time PCR |
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| Uses a computer analyses as it processes. No electrophoresis needed, takes about 10 mins. |
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| Pulse Field Gel Electrophoresis |
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| High molecular weight DNA, is cut with restriction endonuclease. Electrophoresis is done systematically with different angles and currents. Used for epidemiological purposes. HEXAGON. |
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| Advantaged of PFGE |
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| Highly discriminatory - differentiates between strains of the same species. Out break invesigation. Track evolution of organisms. |
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| Disadvantages of PFGE |
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| Equipment is expensive, requires skilled technologist. |
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| Interpreting PFGE |
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| Indistinguishable - banding pattern is the same. Closely Related - 2-3 band differences. Same bug, mutation occurred. Beyond six - not related. |
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| 16s rRNA gene sequencing - premis. |
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| rRNA common to all bacteria. Based on the premis that all bacteria contain 16s ribosomal RNA gene. Its an essential gene. This gene evolves very slowly. Molecular clock – track changes in genes over time using this gene. |
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| Two different areas of the 16s rDNA sequencing |
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| Conserved region - detects bacteria. Divergent - variable within a species. Help to identify the bacteria. Design a primer to target the conserved region. Once you have that amplified, sequence the variable regions which are different from bacteria to bacteria. |
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| How do you identify bactria using 16s rRNA gene sequencing? |
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| PCR primer designed to anneal to conserved regions. Can sequence the variable region and input data into a data base to find what it is analogous to. |
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| 16s Advantages |
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| No need for viable organisms. Can be dead bacteria. Detection and ID of organisms in people who have already started Tx. Detection of slow/non growing organisms. |
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| 16s Disadvantages |
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| Only useful from normally sterile sites - contamination is a big problem because it is so sensitive. Only useful for monomicrobial infections. Slow process. |
