43. Systems for Detection of Pathogens II

Question 1

Molecular Gene Targeting

Answer 1

• Aim to detect a gene or gene products that are pathogen specific
• Nucleic acid amplification techniques (NAAT)
• Polymerase Chain Reaction (PCR)

Question 2

Briefly, describe PCR and qPCR.

Answer 2

PCR

• 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

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

Question 3

Describe Strand displacement amplification (SDA)

Answer 3

This is very similar to PCR. You’re using primers to go along the DNA strand, and it’s producing a fluorescent signal. However, there are slight differences.

SDA relies on a strand-displacing DNA polymerase, typically Bst DNA Polymerase, Large Fragment or Klenow Fragment (3’-5’ exo–), to initiate at nicks created by a strand-limited restriction endonuclease or nicking enzyme at a site contained in a primer. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification.

It is used for N. gonorrhoea and C. trachomatis routinely in the lab.

Make sure not to confuse SDA with PCR.

Question 4

Is a molecular test for one gene good enough?

Answer 4

• Specificity - Is the test unique to the Genus? Species? Type?
• Reliability - Is the target non-essential? transmissible?
• Sensitivity - How many organisms does it take to suggest disease?

• Accuracy - Do we need to detect live organisms?
- Is the detection system susceptible to genomic shifts/mutations?

• Rapidity - Is the result generated going to be beneficial to the patient?

Question 5

How can you have multiple gene targeting?

Answer 5

Microarrays

• 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

Microarrays (tiled arrays)
\+ Covers the whole genome
\+ Strand dependant
\+ Can be used for RNA and Transcriptomics
\+ Can look for microRNA

Microarrays (expression analysis) - you can look at the expression of genes over time

Question 6

Molecular Signatures

Answer 6

• Aim to detect a gene or gene products that are pathogen specific
• Single gene target (PCR) - PCR qPCR
• Multiple gene target (Microarray)
• Mass spectrometry (MALDI-TOF)

Question 7

Bio-signature profiling

Answer 7

• Mass Spectrometry
• MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time-of-Flight)
- Isolate organism
- Lyse with crystalizing matrix
- Ionise and detect time of flight for each particle
•Calculate Mwt (Daltons) for each protein produced

Question 8

Advantages and disadvantages of MALDI TOF profiling

Answer 8

+ Rapid
+ Specific identification

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

Question 9

Biomarkers of virulence. And how would you detect Shiga toxin in E.Coli O157?

Answer 9

• Serotyping
- CSF direct addlutination test
- Serology by ELISA
• Toxin detection

Shiga Toxin detection in E.coli O157

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

Question 10

Advantages and disadvantages of biomarkers of virulence.

Answer 10

+ Good Specificity
+ Good Sensitivity
+ Easily automated

• 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 11

Direct Sequencing

Answer 11

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

Question 12

Molecular detection methods advantages and disadvantages

Answer 12

+ 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

• 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 13

The future for detecting pathogens

Answer 13

• Bio-signature profiling - Following the host transcriptomic profile with microarrays
• Metabolic profiling
• Rapid Intrinsic Fluorescence
• Rapid Point of Care testing
• Lab on a Chip?