MOLECULAR DIAGNOSIS - 7-8 Flashcards
What are conventional testing types?
Microscopy (+/- culture):
- Cheap (minimal equipment and reagents)
- Labour intensive
- Lacks sensitivity/specificity
- Cannot ID cryptic species
- Requires skilled microscopist (in short supply)
- time consuming
Immunology:
- ELISA, IFA, Dipstick ect
- Increased specificity and sensitivity but very variable (as low as 5.5%)
Advantages of molecular testing
- Higher sensitivities and specificities, higher accuracy, and shorter time-to-result
- Suitable for the detection of wide range of pathogens
- eg virus, bacteria, fungus, protozoa ect.
- Multiplexing capability - more information per single test at a lower cost - Can detect fastidious microorganisms and those that cannot be cultured
- Discovery and characterisation of newly emerging or unknown pathogens
- Delays in sample collection, transport, and storage - less effects on the final results
- Suitable for identifying antimicrobial resistance and highly virulent strains
- can be used after the start of antimicrobial therapy 7. Direct analysis of wide range of biological samples
- eg tissues, blood and other fluid - Quantitation
- eg to assess pathogen load in blood, faeces, urine ect - High discriminatory power for microorganisms typing
- eg to track sources of infection, assess transmission and epidemiology studies - Suitable for high throughout analysis of a large number of samples
- Molecular data is easily shared using public databases online
What is PCR
- polymerase chain reaction
- most common molecular assay
Summarise process of PCR
- Denaturing
- Heat DNA to 95 degrees to
overcome hydrogen bonding
between dsDNA
- Double strand —> two single
strands - Annealing
- 72 degrees, polymerase starts making strands. Allow oligonucleotides to hybridise to their complimentary strands - Extension
- Synthesise a new DNA strand at the 3’ end of the primer
- Repeated cycling results in exponential amplification of DNA
What is Quantitative PCR?
- Based on the detection and quantification of fluorescent
reporter - In addition to primers, assay also includes a probe with a
fluorophore and quencher attached - Quencher inhibits fluorscent signal
- Taq polymerase degrades the probe and releases flurophore
- Amount of flurophore released = amount of DNA template present in PCR
- Comparison with standards
- less accurate if standards are not well done
What is Drop digital PCR (dPCR)
- No need for calibration curves
- PCR reaction mixture partitioned into tiny ‘droplets’ micelles (water-in-oil)
- Each droplet is an independent nano-PCR, containing zero, one or more copies of the target nucleic acid
- PCR carried out in each partitioned individually
- Fluorescence of each droplet individually measured
- Defined as positive (presence of PCR product) or negative (absence of PCR product).
- Fraction of positive droplets used to calibrate the target DNA concentration using Poisson statistics
What is reverse transcription PCR (RT-PCR)
- Detection and quantification of RNA -> e.g. RNA virus
- Extract RNA
- Reverse transcriptase transcribes the RNA to complementary DNA (cDNA)
- cDNA is then used as a template for qPCR
what is multiplex qPCR?
- Amplify multiple targets from the same sample
- Requires primer sequences carefully chosen to promote uniformly high amplification efficiency
- Needed to prevent any one assay from dominating
- To compete for the same pool of reaction components
of reaction components - Avoid primer sequences with significant alignment to one another
- Reduces the efficiency and sensitivity of the assay-set
What is Canine distemper virus and how can it be identified
- Serious, often fatal, disease in dogs
- Often, neurological symptoms associated with
canine distemper virus may not be evident for months - Worldwide mortality rate 2nd only to rabies Clinical diagnosis difficult - broad spectrum of clinical signs
- Can present with respiratory signs or enteric signs
- RNA virus so use RT-PCR -> High specificity and sensitivity
What is Bovine Viral Diarrhea virus (BVDV) and how is it identified?
- Costly disease in cattle-> $15-$88 per head
- RNA virus
- Can present with respiratory signs and enteric signs
- Can cause immunosuppressive effect, making them more susceptible to other diseases
- Slow down growth of animals, effect milk production, early death
- Pregnant animal: can cause abortion or the offspring will have a persistent infection and therefore be much more susceptible to disease and die earlier and also be a continuing source of infection to the rest of the herd Causes massive production losses to cattle industry
RT-PCR:
- High specificity and sensiitvity
- Low prevalence situations
- Confirm results of other screening tests
how is chicken sexing done using technology
- Culling males
- Accuracy of vent sexing is ~98%
- 2% margin error = large monetary losses
Sex chromosomes: - ZW in females and ZZ in males
- PCR primers to amplify a region of DNA that differs in size between Z chromosomes in sexes
- 99.9% accurate
What sanger sequencing?
- Sequence DNA
- Widely used - cheap and useful
- Asymmetric PCR
- Di-deoxy bases, 4 colour tags for each bases
- limited to determine the sequence of one fragment of DNA per reaction, up to a maximum length of 700 bases
What is next generation sequencing?
- Can sequence millions of DNA fragment in parallel in one reaction
- Yielding enormous amounts of data
- Does not use sanger method
- Different platforms/different chemistries
- Very high throughput
- Amplicon sequencing - multiple copies of fragments from 1 target gene
- Metagenomics sequencing - short sequence fragments
from ‘all’ DNA
what is next generation sequencing useful for
a) Investigations of disease outbreaks of unknown aetiology
b) Understanding disease transmission and vaccination
requirements
c) Distinguishing vaccination from natural infections
d) Identification of zoonotic pathogens and reservoir hosts
e) Veterinary genomics and precision medicine
f) Understanding resistance to infection
g) Microbiome analysis
