Diagnostic Methods

Question 1

What are some problems with culture-based methods?

Answer 1

Labour intensive
Error prone (fails to discriminate between closely related species)
Insensitive (common swamp rare bacteria)
Some resist in-vitro culture

Question 2

What is specific PCR?

Answer 2

Primers designed to bind to gene or sequence that is specific to particular genus/species

Question 3

What is broad-range PCR?

Answer 3

Primers selected from conserved regions shared by a given taxonomic group

Question 4

Is traditional PCR quicker than culture?

Answer 4

Yes

Question 5

What are possible solutions to contamination?

Answer 5

Physically separate lab areas
Lab coats,
Aerosol-resistant pipette tips

Question 6

When are some contamination risks?

Answer 6

During sample prep
During reaction set up
When products are loaded onto gel for analysis

Question 7

What gel does traditional PCR use?

Answer 7

Agarose gel

Question 8

Which strains of Helicobacter pylori are cagA positive?

Answer 8

4 and 6`

Question 9

What is real time PCR?

Answer 9

Detection of dsDNA by either non-specific binding with fluorescent dyes to amplify product with melt-curve analysis or specific binding with fluorescent probes to amplify product

Question 10

What is reverse transcription PCR?

Answer 10

RNA -> cDNA -> PCR product

DNAse digestion to remove host gDNA

Question 11

What is threshold cycle Ct?

Answer 11

Cycle number at which fluorescence passes threshold value in qPCR

Question 12

How does 16S rRNA work?

Answer 12

16S has conserved regions which the primers bind to and amplify then using bioinformatics (BLAST) to identify

Question 13

What does BLAST stand for?

Answer 13

Basic local alignment search tool

Question 14

What is BLAST?

Answer 14

finds regions of similarity comparing nucleotide/protein sequences calculating an E value of the matches

Question 15

What is multi-locus sequencing typing?

Answer 15

PCR of 7 or more loci comparing it to a database to identify allele type identifying cultures of related genotypes

Question 16

What is whole genome sequencing?

Answer 16

Looks at the whole genome and compares to a database to identify

Question 17

What are the uses of WGS?

Answer 17

Diagnostics
Surveillance
Forensic epidemiology

Question 18

What is nanopore sequencing?

Answer 18

Passes intact DNA strands through protein nanopore, reading sequences in real time as DNA passes through the protein pore

Question 19

What are single nucleotide polymorphisms?

Answer 19

DNA sequence variations that occur when a single nucleotide in genome sequence mutates

Question 20

What is mass spectrometry?

Answer 20

Analytical technique that measures mass of proteins by ionization and measurement of mass-to-charge ratio

Question 21

What is MALDI-TOF?

Answer 21

Matrix-assisted laser desorption/ionization-time of flight
Matrix absorbs UV light and coverts to heat energy charging ions of various sizes attracted to ion detector which is measured

Question 22

What are the limitations of MS for diagnostics?

Answer 22

Needs a colony/culture
Only identify species in database
Does not inform on AR
Not used for viruses