Systems for Detection of Pathogens II Flashcards
What is molecular gene targeting?
What does this technique require?
Detect gene or gene products that are pathogen specific.
Nucleic acid amplification techniques (NAAT).
qPCR
Needs to be specific, reliable, sensitive, accurate and rapid.
What is strand displacement and amplification?
Give 2 examples of things it can detect
Uses primers along target.
Produces fluorescence.
Used for chlamydia and N. gonorrhoea.
Similar to PCR.
What are the 5 main suitable gene targets?
Constitutive.
Virulence.
Antibiotic resistance.
Pathogenic phenotype.
Repetitive.
What is multiple gene targeting?
Microarray.
Ordered short oligonucleotide probes (40-70) attached to slides in defined spots.
Each spot represents a single gene.
Comparative genomic hybridisation used mostly for DNA.
Tiled arrays cover the whole genome and are strand dependant.
Can be used for RNA and transcriptomics and look for microRNA.
What does MALDI-TOF (matrix assisted laser desorption ionization-time-of-flight) do?
What are the advantages and disadvantages?
It isolates the organism, lyses with crystalizing matrix and ionise/detect time of flight for each particle.
Calculate Mwt (Daltons) for each protein produced.
Compare against archives.
Adv- rapid and specific identification.
Dis- requires pure culture, requires rigorous calibration and protocol standardisation, will only identify known profiles.
How can we look for selective genes or gene products that drives the disease process?
What are the advantages and disadvantages?
Latex agglutination tests- uses particles coated with specific antibodies antibody to cell wall antigens.
Specific cell wall antigens are predictive of invasiveness and virulence.
Serotyping- serology by ELISA and CSF direct agglutination test.
Toxin detection- Shiga toxin of E. coli.
Shiga toxin detection in E. coli- enterohaemolysis, agglutination with anti-toxin antibodies and PCR for the presence of the gene.
Adv- good specificity, sensitivity and easily automated.
Dis- not rapid serology (not good for acute infection), some antibodies cross-reactive, virulence inferred by biomarker only in vivo.
Describe NGS
What is the operon and the main problem with using NGS for viruses?
What are the advantages and disadvantages of it?
NGS.
Show difference in single bases.
Operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter.
Not all possible mutations involved in virulence. Silent mutations can be intragenic or synonymous.
Adv- rapid, sensitive (increased in positive samples) and automated (point of care).
Dis- expensive, not for unknowns, expertise needed, labour intensive, contamination, complex methods for nucleic acid extraction and negative samples may still need gold standard culture
What is biosignature profiling?
Biosignature profiling- host transcriptomic profile using microarray.
What is metabolic profiling
Metabolic profiling (MR or gas chromatography for excreted metabolites).
