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Flashcards in Detection & ID of microorganisms Deck (20)
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advantages of molecular-based testing

fastidious organisms or time consuming to isolate
hazardous organisms
high volume tests
more sensitive


specimen collection

avoid contamination
proper equipment
maintain proper time & site of collection


positive control for PCR

DNA prepared from the same site & contains the organism of interest


negative control for PCR detection

DNA prepared from the same site but does not contain the organism of interest


Reagent blank for PCR detection

no DNA present whatsoever


amplification control

omnipresent template unrelated to target used to make sure PCR could show band if organism was present
acts as an internal control to amplify 16S rRNA region that is present in all bacteria


commonly IDed bacteria by molecular methods

Strep pneumoniae
S. agalactiae
Bordetella pertussis
Chlammydia trachomatis/ Neisseria gonorrhea


why detect S. pneumoniae by molecular methods

if in the blood, it can progress very quickly
definitive lab diagnosis is made in 20 minutes


how S. pneumoniae is detected via molecular methods

hypbrid capture assay
no DNA isolation
look for target rRNA sequences
hybrid DNA probe to RNA target
the hybrid that is formed is resistant to the selection reagent


why detect S. agalactiae by molecular methods

recently is normal flora
can lead to post-natal infection of child
all pregnant women are screened & treated if positive
molecular methods are very sensitive!!


why detect C. difficile by molecular methods

difficult to culture


determining normal flora vs active infection of C. difficile

if the PCR amplification products cross the threshold earlier than the cutoff its an active infection
later is normal flora


Chlamydia trachomatis/Neisseria gonorrhea (CTNG) testing by molecular methods

use the same assay for both organisms
not normal flora & any signal is an infection
can't use for legal cases
high volume testing


Bordetella pertussis by molecular methods

nasopharyngeal swab, very difficult to culture


why detect mycobacterium by molecular methods

take up to 5 weeks to grow
very difficult to speciate


how mycobacterium is detected by molecular methods

fluorescent probe assay (PNA-FISH)
PNA probe (protein-nucleic acid)
very stable
binds tighter than a regular nucleic acid probee


Candida detection by molecular methods

very difficult to speciate w/ biochemical methods
PNA to the rRNA


why use molecular methods for viral detection

shorter detection window
fast turn around time
perform viral load assays
high volume


hep C viral genotyping

used to determine genotype which can determine disease prognosis & response to treatment
real time quantitative RT-PCR
3 PCR reactions to cover important genotypes


HIV viral genotyping

HIV genes mutate readily
HIV gene mutations are detected by directly sequencing the protease & reverse transcriptase genes