Systems for Detection of Pathogens II Flashcards
What is molecular gene targeting?
β detecting a gene of gene products that are pathogen specific
What are the two techniques used for molecular gene targeting?
β NAAT
β PCR
How does PCR work?
β Two DNA primers specific for opposite strands are used to amplify the DNA region
β The product is visualised by fluorescent tags or staining in gels for an amplicon of an exact size
What does quantitative PCR measure?
β the speed at which a PCR amplicon product accumulates by the amount of fluorescence released
What two conditions is strand displacement amplification used for?
β gonorrhoea
β chlamydia
Which gene is a suitable target for chlamydia and why?
β IS711
β there are multiple copies of the same gene in every chlamydia
Why do you need more copies of H.Pylori than chlamydia to get a positive test result?
β there is only one copy of the target gene
What 5 variables need to be taken into account when doing a molecular test?
β Specificity β Reliability β Sensitivity β Accuracy β Rapidity
What is an instant bedside test useful for?
β Diagnosis of pediatric meningitis
What is a microarray?
β Ordered short nucleotide probes (40-70) attached to slides in defined spots
β each spot represents a single gene
How is comparative genomic hybridisation done?
β sample DNA and control DNA are fluorescently labelled
β they are both hybridised onto a microarray
What are the 4 advantages of tiled arrays?
β Covers the whole genome
β strand dependent
β can be used for RNA and transcriptomics
β can look for microRNA
What are the 3 ways to detect molecular signatures?
β Single gene target
β multiple gene target
β mass spectrometry
How does mass spectrometry (MALDI-TOF) work?
β Isolate organism
β Lyse with crystallizing matrixx
β Ionise and detect time of flight for each particle
β Calculate molecular weight (daltons) for each protein produced
What are the two advantages of mass spectrometry?
β Rapid
β Specific identification
What are the three disadvantages of mass spectrometry?
β Requires a pure culture
βRequires rigorous calibration and protocol standardisation
β will only identify known profiles
What does the latex agglutination test use?
β Uses particles coated with specific antibody to cell wall antigens
How do you detect the Shiga toxin in E.Coli O157?
1) enterohaemolysis
2) agglutination with anti-toxin antibodies
3) PCR for the presence of the gene
What 3 pathogens do you use a CSF direct agglutination test for?
β Neisseria Meningitidis B
β Haemophilus influenzae type B
β Streptococcus pneumoniae
What is targeted when looking at biomarkers of virulence?
β looking for selected genes that drive the disease process
β use proteins that are inside the bacteria or cell membrane components
What are the 5 disadvantages of biomarkers?
β Serological response is not rapid therefore not useful in acute infections
β Single sera results are meaningless due to possible previous exposure
β some antibodies are cross-reactive
βvirulence is only inferred by the presence of a biomarker
β only in vivo testing of a cultured pathogen infected into an animal can prove virulence
What can sequencing show?
β Differences in single bases in strains or resistance mutations to antibiotics
What are the 5 advantages of molecular detection methods?
β Rapid
β faster detection of pathogens than traditional techniques
β allows appropriate timely antimicrobial therapy and infection control interventions
β increased sensitivity over culture and microscopy based techniques in positive samples
β can be automated
What are the 8 disadvantages of molecular detection methods?
β Expensive β does not screen for unknowns β requires expertise β labour intensive β possibility of contamination β requires complex and efficient methods of extraction of nucleic acids β negative samples may still need gold standard culture β hospital cost
What is bio signature profiling?
β taking people who have the active disease and seeing which genes are switched on when the disease is present
What kind of genes would be switched on during disease?
β ferritin
β cytokines
β antibody genes
What is metabolic profiling?
β using excreted metabolites to diagnose malaria in urine
What is the advantage of point of care testing?
β rapid sequencing of samples direct at the bedside
What are 5 things that a new system for detecting pathogens has to have?
β Reliable, sensitive,specific and rapid
β applied to correct specimen
β must derive from a large reference database
β constantly updates with new species of variants
β must be as good as or better than gold standard
