Systems of Pathogen Detection II Flashcards
What is the aim of molecular gene targeting?
Aim to detect a gene or gene products that are pathogen specific
What are the 2 molecular gene targeting techniques
- Nucleic acid amplification techniques (NAAT)
- Polymerase Chain Reaction (PCR)
What is PCR?
PCR is amplifying DNA specifically using primers / oligonucleotides in a cyclical process viewed using banding on gel electrophoresis or fluorescence
How is product amplified in PCR?
Two DNA primers (18-20bp) specific for opposite DNA strands, used to amplify DNA region
How is the product visualised in PCR?
Product is visualised by fluorescent tags or staining in gels for an amplicon of an exact size
Which pathogens are detecting using NAAT and PCR?
- Influenza/H1N1
- Norovirus
- MRSA
- HIV
- Hepatitis B
- Hepatitis C
- Mycobacterium tuberculosis
- CMV
- EBV
What is the role of Quantitative PCR?
Measures the speed at which a PCR amplicon product accumulates by the amount of fluorescence released
What is the advantage of using PCR?
PCR provides specificity and a good approximation of quantitation
What is SDA?
Strand Displacement Application (SDA) is similar to PCR; uses primers and fluorescence as well
Which pathogens are identified using SDA?
Used to identify N. gonorrhoea and C. trachomatis
Which genes are suitable targets for pathogen detection?
- Constitutive
- Virulence
- Antibiotic resistance
- Pathogenic phenotype
- Repetitive
How is specificty of a test determined?
Is the test unique to the Genus
Species
Type
How do we determine if the test is reliable?
If the test tells us if the target is:
- non-essential
- transmissible
How do we know how sensitive a molecular test is?
By the no. of organisms it takes to suggest disease
- for every sample type
- for every host type
- for every epidemiological niche
What factors determine the accuracy of a molecular test?
- Detecting live organisms
- Susceptibility of detection system to genomic shifts/mutations
Why is the rapidity of a molecular test significant?
Is result generated going to be beneficial to patient?
Instant Bedside? - Diagnosis of paediatric meningitis
Same Day? - Transmission/Quarantine
Next Day? - Antibiotic resistance
Next Week? - Chronic persistent infections
How is multiple gene targeting conducted?
Microarrays
Comparative Genomic Hybridisation (CGH) used mostly for DNA
Outline how microarrays are used to detect multiple genes
Ordered short oligonucleotide probes (40-70mer) attached to slides in defined spots.
Each spot represents a single gene
What info does a microarray provide?
Can look for genes and their expression
Some genes may be present but not expressed
What are the advantages of using microarrays?
- Covers whole genome
- Strand dependant
- Can be used for RNA and Transcriptomics
- Can look for microRNA
What is the use of Microarray expression analysis?
Can identify genes over time and how certain expressions change over time
- Some genes may be present to start off with but lost later on
What is the purpose of molecular signatures?
Aim to detect a gene or gene products that are pathogen specific
Outline single gene target signatures
Single gene target (PCR)
- PCR
- qPCR
Outline the multiple gene target signatures
Microarray
What is the use of Mass Spec?
Identifies different components of the organism (not just DNA)
What is Mass Spec MALDI-TOF?
Mass Spectrometry MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time-of-Flight)
Outline how mass spec MALDI-TOF is conducted
- Isolate organism
- Lyse with crystalizing matrix
- Ionise
- Detect time of flight for each particle
- Calculate Mwt for each protein produced
- Compare against database
What does each peak represent in mass spec?
Each peak is specific for small peptide fragments
How does mass spec identify a pathogen?
Different organisms produce specific patterns that are compared with known pathogen patterns to identify which it is
Outline the advantages of using MALDI-TOF
- Rapid
- Specific Identification
- Automated
What are the disadvantgaes of using MALDI-TOF mass spec?
- Requires pure culture
- Requires rigorous calibration and protocol standardisation
- Will only identify known profiles
Define biomarkers of virulence
Selected genes or gene products that drive the disease process
Outline biomarkers of virulence used
We use biomarkers of bacterial cell components e.g. O-antigens, peptidoglycan etc.
How does latex agglutination test detect the virus?
Latex agglutination test uses particles coated with specific antibody to cell wall antigens
CSF Direct Agglutination test
Which viruses are detected using an agglutination test?
Neisseria meningitidis Group B Haemophilus influenzae type b Streptoccoccus pneumoniae type 3
What do cell wall antigens tell us about a virus?
Specific cell wall antigens are predictive of invasiveness and virulence
How is the Shiga toxin of E.coli 0157 detected?
1) Enterohaemolysis
2) Agglutination with anti-toxin antibodies
3) PCR for the presence of the gene
What are the advantages of the 0157 Stx detection method?
- Good Specificity
- Good Sensitivity
- Easily automated
What are the disadvantages of the E.coli Type O157 detection approach?
- Serological response is slow; not useful in acute infections
- Single sera results are meaningless as possible previous exposure
- Some antibodies cross-reactive
- Virulence only INFERRED by presence of a biomarker
ONLY in vivo testing of cultured pathogen infected into an animal model can prove virulence
How is rapid sequencing conducted?
Rather than looking at the assembled product we look at ALL the pieces individually
What is the benefit of using direct sequencing to identify pathogens?
Sequencing can show differences between SINGLE bases in strains Or resistance mutations to antibiotics
What are the 2 types of possible silent mutations?
- Intragenic (between genes)
- Synonymous (not altering coding)
Outline the advantages of molecular detection methods
- Rapid
- Faster detection than traditional techniques
- Allows appropriate, timely antimicrobial therapy and infection control interventions
- Increased sensitivity over culture + microscopy based techniques in +ve samples
- Automated and has potential for Point of Care testing
Outline disadvantages of molecular detection methods
- Expensive
Doesn’t screen UNKNOWNS - Requires expertise
- Labour intensive
- Contamination
- Require complex + efficient methods for nucleic acid extraction
- NEGATIVE samples may STILL need Gold Standard culture
What are the future techniques of pathogen detection?
- Bio-signature profiling
- Metabolic profiling
- Rapid Intrinsic Fluorescence
- Rapid Point of Care Testing
What does a biosignature profile show us?
Following the host transcriptomic profile with microarrays
During active disease can identify which genes are turned ON to create profile
