Molecular Methods for Diagnosis Flashcards
traditional methods
culture- takes a long time, cant grow everything in lab
staining- insensitive- requires more than 100,000/mL
serology- individual must make immune response, which takes time. also doesnt occur in immunosuppressed individuals
advantages of molecular amplification
1 can detect very small numbers
2 theoretically fast
3 can detect organisms that cannot be cultured
4 can be miultiplexed to detect more than one target simultaneously
disadvantages of molecular amplification
1 must know specific NA sequence and follow potential changes
2 must be able to interpret meaning of detecting specific sequence
3 must be able to exclude false positives (contamination)
4 present technology may not allow theoretical speed of test- must be batched
amplification techniques commonly used
PCR
TMA (transcription mediated amplification)
LCR (ligase chain reaction)
bDNA (branched chain DNA amplification)
qualitative formats
uses amplifiation to detect the smallest possible number of target molecules
most sensitive, but only useful when detection of the organisms correlates w/ disease
more common
quantitative
generates a graded signal that correlates w/ higher target number (HIV load, CMV, herpes)
levels of development for molecular tests
“user developed”- requires complete validation of test characteristics and specimens
“analyte specific reagent”- validated when developed in lab but reagents need only routine QC
FDA cleared- requires in lab verification for cleared specimen types
Research Use Only- not reimbursed
FDA cleared molecular assays
MRSA chlamydia, gonorrhea, trichomonas clostridium difficile multiplex assay for bacterial sepsis pathogens multiplex assay for CNS pathogens
molecular diagnosis of TB
sample from colony- FDA approved RNA/DNA hybridization
sample from specimen: FDA approved direct test- TMA method
test accuracy decreases depending on the type of sample ( smear positive vs smear negative)
acid fast bacteria in fixed tissue- lab developed, variable results
multiplex arrays
amplification of nucleic acid targets
capable of detecting multiple agents- most common
blood sample must be allowed to grow bacteria before use in multiplex array
epidemiolgy uses
w/ outbreak, can sequence bacteria to see if they come from the sample place
agreed upon standards for how much genetic info can differ before it is considered the same
problems w/ whole sequence analysis
too much data- no agreed upon standards about which differences matter
identifying difficult to grow bacteria
done by using 16 S rRNA gene amplification
conservation of rRNA active sites allows primer development
variability in non critical regions allow comparisons of organisms
RNA gene sequencing allows for description of microbial communities in normal or diseased states
ex. lung in cystic fibrosis
mass spectrometry
proteomic/metabolomic approac to identification of microorangisms
identification of signatures associated w/ pathogens by breaking them apart and watching flight patterns
