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12 Cards in this Set

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What are the TRADITIONAL TECHNIQUES used to identify pathogens?
1) CULTURE
2) STAINING
3) SEROLOGY
CULTURE?
- this relies on being able to grow and identify a pathogen, but it takes days and weeks- - BUT there are things that we cannot grow or identify after its been grown
STAINING?
- this relies on the reaction of dyes with various components of the pathogen; and most stains require more than 100,000 organisms/ ml of specimen to detect any by microscopy

- some techniques (ie. immunofluorescence) can detect less tha this but are still relatively insensitive
SEROLOGY?
- this relies on the infected individual making an immune response to the pathogen

- it usually takes weeks, and it may not occur in immunosuppressed individuals
What does MOLECULAR DETECTION require and what are 2 examples of it?
- Molecular detection requires knowledge of the DNA of a pathogen

1) HYBRIDIZATION: has been used but is insensitive

2) AMPLIFICATION: allows a million-fold multiplication of the target sequence or of a specific detector molecule
What are some ADVANTAGES of molecular method?
1) can detect very small numbers of organisms

2) theoretically fast

3) can detect organisms that cannot be cultured
What are some DISADVANTAGES of molecular method?
1) must have specific nucleic acid sequence

2) must be able to interpret the meaning of detecting the specific sequence

3) must be able to exclude false positives, most frequently from amplicon contamination of environment of lab

4) present technology might not allow theoretical speed of tests to be batched- - so may take days
What are the common techniques used today?
1) PCR
2) TMA (TRANSCRIPTION MEDIATED AMPLIFICATION)
3) LCR (LIGASE CHAIN REACTION)
4) bDNA (branched CHAIN DNA AMPLIFICATION)
What are the formats of observatons used? (2)
1) QUALITATIVE: this uses amplification to detect the smallest possible number of target molecules.

it's most sensitive, but may be useful only when detection of any evidence of the organism correlates with disease

2) QUANTITATIVE: this technique generates a graded signal that correlates with target number.

- it's useful in following therapy

- may also be useful in trying to sort out endogenous carriage from disease
SEE EXAMPLES OF VIRUSES, BACTERIA, FUNGI, AND PROTOZOA detected by molecular techniques.
*
What's an example of a molecular method testing for disease?
CHLAMYDIA/ GC by TMA
What are other molecular techniques used and examples?
IDENTIFICATION OF ORGANISM:
1) HYBRIDIZATION
2) PCR and SEQUENCING
ex) for id of an unknown organism

STRAIN COMPARISON FOR EPIDEMIOLOGY
1) PFGE (pulsed field gel electrophoresis)
2) RFLP and other fragment identification schemes
ex) mysterious bacteremia in dialysis patients