Detection of environmental pathogens

Question 1

Benefits of bacterial culture

Answer 1

CFU counts
Phenotypic traits
Pathogenicity
Biomass
Culturable means the bacteria is viable

Question 2

Limitations of bacterial culture

Answer 2

Hazardous densities of pathogenic bacteria
Not representative of diversity or proportionality (no media is representative for all organisms)
Underestimation of number
Bacteria can enter viable but non-culturable (VBNC) state

Question 3

Molecular techniques

Answer 3

Overcomes VBNC issue
Allows all or targeted populations to be observed
Whole cell based or nucleic acid based
Does not determine whether bacteria are live or dead

Question 4

Nucleic acid based detection of bacteria

Answer 4

Extraction of nucleic acids
Polymerase chain reaction (end point, real time, qPCR)
DNA sequencing (Sanger, next generation)

Question 5

Nucleic acid extraction

Answer 5

Cell lysis and buffering
Separation of nucleic acid from cell debris (binding)
Elution of nucleic acid in pure form

Question 6

PCR

Answer 6

Requires prior sequence knowledge for oligonucleotide primers

Polymerisation using thermostable enzyme (Taq polymerase) from Thermus aquaticus

Question 7

End point PCR

Answer 7

Not quantitative
Products visualised by gel electrophoresis
Confirmed by DNA hybridisation (southern blot)

Question 8

16s rRNA

Answer 8

Highly conserved and unique to bacteria and bacteriophages

Conserved regions used to design primers (not whole gene)

Question 9

Limitations of end point PCR

Answer 9

Inhibition (Fumic acid)
Low sensitivity 
Short dynamic range
Low resolution
Only discriminated by size
Non-automated

Question 10

Real-time PCR

Answer 10

Fluorescent labels (Taqman hydrolysis probes or dsDNA binding dyes) to detect amplification in real time
Can detect multiple targets at once
Quantification without culturing

Question 11

SYBR and hydrolysis probes

Answer 11

Quenched SYBR green dye in solution.
Incorporates into dsDNA and releases fluorescence
Increased fluorescence with amplification
Hydrolysis probes have fluorescence and quencher.
Primer anneals and probe hybridises. Exonuclease releases fluorescence

Question 12

HRM RT PCR

Answer 12

High resolution melting

Detects sequence differences based on melting curve

Question 13

Disadvantages of qPCR

Answer 13

Also affected by inhibitors
More optimisation required than end point
Does not resolve live/dead issue

Question 14

dPCR

Answer 14

One chip at a time, one sample at a time
Increased resolution
No inhibition
Low throughput

Question 15

Sanger sequencing

Answer 15

Uses dideoxynucleotides (ddNTPs) rather than deoxynucleotides (dNTPs)
ddNTPs incorporated to stop the fragment