Systems for Detection of Pathogens II

Question 1

Systems for the detecting pathogens - list 5

Answer 1

• Gene targeting
• Genome targeting
• Biomarkers of virulence
• Rapid sequencing
• Future Directions

Question 2

Molecular Gene Targeting - aim and 2 methods

Answer 2

Aim to detect a gene or gene products that are pathogen specific

Nucleic acid amplification techniques (NAAT)
Polymerase Chain Reaction (PCR)

Question 3

Polymerase Chain Reaction - brief method

Answer 3

Two DNA primers (18-20bp) specific for opposite
DNA strands, used to amplify
DNA region
Product is visualised by fluorescent tags or
staining in gels for an amplicon of an exact size

Question 4

Quantitative PCR (qPCR) - purpose

Answer 4

Measures the speed at which a PCR amplicon
product accumulates by the amount of
fluorescence released

Question 5

Multiple gene targeting (Microarrays) - brief method + what is used for DNA mostly

Answer 5

Ordered short oligonucleotide probes (40-70mer) attached to slides in defined
spots.
Each spot represents a single gene
Comparative Genomic Hybridisation (CGH) used mostly for DNA

Question 6

Microarrays (Tiled arrays) - advantages

Answer 6

Advantages
Covers the whole genome
Strand dependant
Can be used for RNA and Transcriptomics
Can look for microRNA

Question 7

Molecular Signatures - aim and give examples of methods

Answer 7

Aim to detect a gene or gene products that are pathogen specific

Single gene target (PCR)
PCR
qPCR
Multiple gene target (Microarray
Microarray
Mass spectrometry (MALDI-TOF)

Question 8

Mass Spectrometry - brief method

Answer 8

Isolate organism
Lyse with crystalizing matrix
Ionise and detect time of flight for each particle

Compare against an archival database, 62,500 unique spectral profiles
Identifying 1,160 species and 233 genera

Question 9

MALDI TOF profiling - advantages

Answer 9

Advantages
Rapid
Specific Identification

Question 10

MALDI TOF profiling - disadvantages

Answer 10

Disadvantages
Requires pure culture
Requires rigorous calibration and protocol standardisation
Will only identify known profiles

Question 11

Biomarkers of Virulence - purpose

Answer 11

Looking for selected genes or gene products that drive the disease process

Question 12

Biomarkers of virulence - how are they used

Answer 12

Latex agglutination test
uses particles coated with specific antibody to cell wall antigens
CSF Direct Agglutination test

Question 13

Shiga Toxin detection in E.coli O157 - brief method

Answer 13

Shiga Toxin detection in E.coli O157

1) Enterohaemolysis
2) Agglutination with anti-toxin antibodies
3) PCR for the presence of the gene

Question 14

Biomarkers of virulence - advantages

Answer 14

Advantages
Good Specificity
Good Sensitivity
Easily automated

Question 15

Biomarkers of virulence - disadvantages

Answer 15

Disadvantages:

Serological response is not rapid
therefore not useful in acute infections
Single sera results are meaningless
due to possible previous exposure
Some antibodies are cross-reactive
Virulence is only INFERRED by the presence of a biomarker
ONLY in vivo testing of cultured pathogen
infected into an animal model can prove virulence

Question 16

Sequencing shows what

Answer 16

Sequencing can show differences between SINGLE bases in strains
Or resistance mutations to antibiotics

Question 17

Silent mutations can be what

Answer 17

Silent mutations can be
Intragenic (between genes)
or
Synonymous (not altering coding)

Question 18

Molecular detection methods - advantages

Answer 18

ADVANTAGES:

Rapid.
Faster detection of pathogens than traditional techniques.
Allows appropriate, timely antimicrobial therapy and infection
control interventions
Increased sensitivity over culture and microscopy based
techniques in POSITIVE samples
Can be automated and has potential for Point of Care testing

Question 19

Molecular detection methods - disadvantages

Answer 19

DISADVANTAGES
Expensive
Does not screen for UNKNOWNS
Requires expertise
Labour intensive
Possibility of contamination
Require complex and efficient methods for extraction of nucleic acid
NEGATIVE samples may STILL need Gold Standard culture

Question 20

Explain they need for a gold standard culture in molecular detection methods

Answer 20

NEGATIVE samples may STILL need Gold Standard culture
Hospitalization costs accounted for 95% of health-system costs
among patients suspected of tuberculosis. In culture-negative
patients, PCR tests do not significantly decrease time to tuberculosis
exclusion.