Systems for Pathogens 2

Question 1

What is molecular gene targetting

Answer 1

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

Question 2

How can we amplify DNA

Answer 2

• NAAT
• PCR

Question 3

Briefly explain how PCR works

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
• Primer can also amplify wanted bit of DNA

Question 4

Briefly explain what qPCR does

Answer 4

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

Question 5

What is Strand displacement amplifacation

Answer 5

A similar DNA amplification technique like PCR

Question 6

What genes are suitable targets for PCR

Answer 6

• Constitutive
• Virulence
• Antibiotic resistance
• Pathogenic phenotype
• Repetitive

Question 7

How can we ensure if molecular tests for one gene is good enough

Answer 7

Is the test… for its use case

• Specific
• Reliable
• Sensitive
• Accurate
• Rapid

Question 8

What can we use to detect single gene targets

Answer 8

• PCR
• qPCR

Question 9

What is used to detect organism composition

Answer 9

Mass spectrometry (MALDI-TOF)

Question 10

How does MALDI TOF profiling work

Answer 10

• Isolate organism
• Lyse with crystallising matrix
• Ionise and detect the time of flight for each particle

Question 11

How is data from MALDI TOF used

Answer 11

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

Question 12

What are the advantages of using MALDI TOF profiling

Answer 12

• Rapid
• Specific Indentifacation

Question 13

What are the disadvantages of using MALDI TOF profiling

Answer 13

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

Question 14

What are biomarkers of virulence

Answer 14

Specific cell wall antigens that are predictive of invasiveness and virulence

Question 15

How can we test for these virulence markers

Answer 15

• CSF Direct Agglutination test
• Latex particles coated with specific antibodies to cell wall antigens

Question 16

What are the advantages of using biomarkers of virulence

Answer 16

• Good specificity
• Good sensitivity
• Easily automated

Question 17

What are the disadvantages of using biomarkers of virulence

Answer 17

• 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 18

How can direct sequencing be used

Answer 18

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

Question 19

What are the advantages in using molecular detection methods

Answer 19

• 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 20

What are the disadvantages in using molecular detection methods

Answer 20

• 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 21

What are the future detection techniques for pathogens

Answer 21

• Bio-signature profiling
• Metabolic profiling
• Rapid point of care testing
• Lab on chip