SP2

Question 1

What are the two molecular genetic gene targeting for pathogens?

Answer 1

→Nucleic acid amplification techniques (NAAT)

→Polymerase Chain Reaction (PCR)

Question 2

How long are DNA primers?

Answer 2

→18-20bp

Question 3

What is qPCR?

Answer 3

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

Question 4

What is strand displacement amplification?

Answer 4

→Relies on astrand displacement DNA polymeraseand a DNA nicking event targeted via primer design and anicking endonuclease

Question 5

Which genes are suitable targets for for molecular testing for pathogens?

Answer 5

→Antibiotic resistance →Pathogenic phenotype Repetitive

Question 6

What pathogens can be detected using strand displacement amplification?

Answer 6

→Neisseria meningitidis- genital, IS711

→Trophyrema whipplei

Question 7

Examples of pathogens detected using PCR

Answer 7

→Respiratory Syncytial →Virus Influenza A / B

→Hepatitis B & C →Coronavirus →Adenovirus →Cytomegalovirus →Epstein Barr Virus

Question 8

What factors need to be considered to decide whether molecular test for one gene is good eneough?

Answer 8

→specificity
→reliability
→sensitivity
→accuracy
→rapidity

Question 9

What is MALDI-TOF?

Answer 9

→Matrix Assisted Laser Desorption Ionization-Time-of-Flight

Question 10

Describe the test for MALDI-TOF

Answer 10

→Isolate organism

→Lyse with crystalizing matrix

→Ionise and detect time of flight for each particle

→Calculate Mwt (Daltons) for each protein produced

→Compare against an archival database

Question 11

What are the advantages of MALDI-TOF?

Answer 11

→Rapid

→Specific Identification

Question 12

What are the disadvantages of MALDI-TOF?

Answer 12

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

Question 13

What type of test can be done for biomarkers of virulence?

Answer 13

→serotyping

Question 14

What are biomarkers of virulence for E.coli?

Answer 14

→Enterohaemolysis
→Agglutination with anti-toxin antibodies
→PCR for the presence of the gene

Question 15

What are the advantages of biomarkers of virulence?

Answer 15

→Good Specificity →Good Sensitivity →Easily automated

Question 16

What are the disadvantages of biomarkers for virulence by serotyping?

Answer 16

→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 17

Why look at the assembled product when you can look at ALL the pieces?

Answer 17

→Rapid Next Generation Sequencing

Question 18

What are the advantages of molecular detection methods?

Answer 18

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

What are the disadvantages of molecular detection methods?

Answer 19

→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

What is the future of molecular testing?

Answer 20

→Following the host transcriptomic profile with microarrays

→metabolic profiling

Question 21

Give two examples of metabolic profiling

Answer 21

→urine after malaria infection using nuclear magnetic resonance
for excreted metabolites
→Breath urine after tuberculosis infection using gas chromatography
for volatile organic compounds

Question 22

Examples of point of care testing

Answer 22

→Lab on a Chip →Nanotechnology

→Microfuidics