Diagnostics Flashcards
PCR
usually viruses
amplify region of interest in genome
number of copies provides information of presence of gene of interest in starting material
stages of PCR
Denaturation - high temp - 94 - break hydrogen bonds between base pairs
Annealing - cool down - 50-60 - bind bases to oligonucleotide primers
Extension - heated - 74 - synthesise new DNA strand from 3’ end
Cycling - repeated multiple times - around 40
semi quantitive PCR
Amount of end product visualised using gel electrophoresis
visual confirmation of presence of areas of interest
quantitative PCR (qPCR)
flourescent dye or target specific probe binds to area of interest
level of flourescence measured with flourometer
reverse transcriptase PCR (RT-PCR)
use reverse transcriptase to produce DNA from RNA template
used as template for normal PCR
molecular diagnostics
direct detection of pathogen DNA from start of infection
early diagnosis
swabs from animal or environment
then pCR used
benefits of PCR
high specificity, high sensitivity
rapid turnaround
no risk of overculturing
disadvantages of PCR
low positive predictive value - may still be positive after and infection cleared
varying sensitivity
needs specialist equipment
PCR inhibitors - cause false negatives - bile salts, polysaccharides in feces, haem, glycogen, fats, proteinases in milk, urea
ELISA
detect antigens or antibodies in a blood sample
antibodies may remain after infection
direct - antibodies used to test for antigen
indirect - antigen used to test for antibodies
bacteria and bacterial toxins, viruses, protozoa, antibodies to any of these or parasites or yeasts
ELISA process
antigen or antibody in well
enzyme labelled antibody or antigen added
bind
enzyme specific marking substrate added
reaction takes place - colour or flourescence
sandwich ELISA
Capture antibody (binds to any antigen) –> antigen –> specific antibody
competitive ELISA
sample and reference substance added at the same time
analyte concentration measured and compared
IHC
uses antibodies to detect antigen in a tissue on histology
monoclonal antibodies - produced by same line of immune cells
polyclonal antibodies - derived from different lines of cells
differentiate between inflammation and neoplasia, and to find out what line of cells a neoplasia is (what type of cancer)
indications for kidney biopsy
proteinuria - damage to glomerular filter lets more protein into nephron, can see damage to endothelium or podocytes
acute renal failure
chronic kidney disease
renal azotemia that can’t be categorised as acute or chronic renal failure
immune mediated glomerularnephritis
immune complexes deposited in glomerulus –> formation of antibodies against glomerular basement membrane –>a activation of inflammatory cascade
any condition that stimulates immune system for a long time
thicken walls - because of deposition of antibody-antigen comlplexes
destruction of podocytes
more protein through because filter damaged
