Quiz 3 Content Flashcards
1
Q
Why detect foodborne pathogens? [2]
A
- Public health concern - Health Canada estimated that approximately 13 million Canadians suffer from illness caused by foodborne bacteria every year
- Economic concern - The annual cost related to these illnesses and related deaths are between 12 and 14 billion dollars
1
Q
Describe traditional culture-dependent pathogen detection.
A
- Prepare a 1:10 dilution and incubate for 24 hours in a non-selective enrichment (give injured/stressed bacteria a chance to recover; also if very few pathogenic bacteria are present, allow them to grow to detectable numbers)
- Incubate in selective enrichment for 24 hours
- Streak onto differential/selective media and incubate for another 24 hours
- Biochemical analysis of suspected pathogenic colonies will follow
- This method takes 48 to 72 hours for preliminary results
2
Q
Describe the isolation of E.coli O157:H7 using traditional culture-dependent detection.
A
-
Tellurite Cefixime - Sorbitol MacConkey Agar
- The sorbitol-containing medium differentiates nonsorbitol fermenting O157:H7 STEC colonies from those of commensal strains (i.e., sorbitol fermenters)
- Cefixime inhibits the growth of other enteric bacteria
- Potassium tellurite inhibts non-O157 STEC
3
Q
How is Tellurite Cefixime - Sorbitol MacConkey Agar used to isolate E. coli O157:H7? [3]
A
- Cifixime inhibits other enteric bacteria
- Potassium tellurite inhibits non-O157 STEC
- The sorbitol containing medium differentiates nonsorbitol fermenters from sorbitol fermenters
4
Q
Describe the isolation of Salmonella using traditional culture-dependent methods.
A
-
Xylose-lysine deoxycholate (XLD) agar
- Selective agar, inhibits growth of Gram-positive microbes
- Salmonella appears black (H2S production)
- E.coli appears yellow after 18-24 hours of incubation at 37C
5
Q
What biochemical analysis generally follows the traditional culture based method?
A
- Serotyping (based on the slide agglutination test)
6
Q
What is serotyping?
A
- A mode of further classification based on two types of antigens:
- O antigen: outermost portion of the lipopolysaccharide
- H antigen: flagellar protein (Phase 1/Phase 2 flagella
7
Q
What is the O antigen?
A
Outermost portion of the lipopolysaccharide
Somatic antigens
8
Q
What is the H antigen?
A
Flagellar protein
Phase 1/Phase 2 flagella
9
Q
What is the slide agglutination test?
A
- Antigens are assayed with antibodies on an inert surface
10
Q
Describe the isolation of Campylobacter.
A
-
Campylobacter blood free selective agar (CCDA)
- Charcoal Cefoperazone Deoxycholate
- Defoperazone and Amphotericin B were used as supplements for selective growth of Campylobacter
11
Q
Describe the isolation of Listeria monocytogenes.
A
- In early studies - incubation for prolonged periods at 4C on agar plates until the formation of visible colonies (i.e., very few pathogens can grow at this low temperature; however, this can take over a month for colonies to grow) - selective agars were developed to overcome this.
- Selective agar (selective inhibitory components; indicator = aesculin & ferrous iron)
- L. monocytogenes hydrolyses aesculin, producing black zones around the colonies due to the formation of black iron phenolic compounds
12
Q
What are the advantages of the traditional culture-dependent isolation methods? [2]
A
- Sensitive (can detect pathogens even in trace amounts)
- Reliable (still represent the gold standard)
13
Q
What are the disadvantages of the traditional culture-dependent isolation methods? [2]
A
- Time consuming (at least 3 days required for results)
- Labour intensive
14
Q
Why are rapid detection methods needed? [3]
A
- Detection results are desired within a shorter period of time
- Some foods are perishable
- Require special storage conditions
15
Q
Describe the workflow of rapid detection methods.
A
- Dilute and enrich for 10-28 hours
- Prepare sample
- Conduct assay
16
Q
What are the targets of current rapid detection technologies?
A
- ELFA & bacteriophage-based biosensor target surface molecules
- PCR and other molecular detection systems target DNA and ribosomes (inside the cell)
17
Q
Describe the history of detection of Salmonella and L. monocytogenes.
A
18
Q
How does ELISA work?
A
- Antigens coated onto ELISA plate
- Serum/sample containing primary antibodies is added
- Non-antigen binding antibodies are washed off the plate
- Secondary antibody-conjugated with an enzyme is added
- Excess secondary antibody is washed off the plate
- Substrate for the enzyme (chromagen) is added
- Enzyme reacts with the substrated producing colour - intensity of the colour correlates with the level of antigen
19
Q
Describe ELFA.
A
- First described in 1979
- First introduced to food industry in the early 90s
- Similar to ELISA, but ~100 times more sensitive for reporting results
- Utilizes a substrate that yields a fluorescent, rather than visible, product on interaction with the immunoglobulin-linked enzyme
Enzyme-linked fluorescent assay
20
Q
What are the steps in PCR?
A
- Denaturing (95°C)
- Annealing (40-60°C)
- Extension (72°C)
21
Q
Describe real-time PCR: The Taqman Probe Method
A
- The primers bind to a genetic sequence on the ssDNA; the probe also binds to a region of the sequence.
- Probes contain fluorescent reporter dye at one end, whose signal is absorbed by the quencher dye at the other end.
- As the DNA enzyme extends the primer, it breaks the probe apart. This separates the reporter dye from the quencher, which increases the reporter signal.
- Successive heating and cooling cycles allow the DNA fragments to replicate exponentially.
- A separate detection phase is not necessary because signal is measured and analyzed at the end of each cycle during amplification
22
Q
Why isn’t a detection phase necessary in Real-time PCR?
A
- Signal is measured and analyzed at the end of each cycle during amplifcation
23
Q
What are the advantages of PCR? [4]
A
- Adaptable to new targets (can always design new primers and probes)
- Potential for high specificity
- Good multiplexing (multiple primers may be designed)
- Rapid & sensitive
25
Q
What is LAMP?
A
- A novel nucleic acid amplification method developed by Notomi et al. (2000).
- 4 primers comprising 2 inner primers and 2 outer primers are used to target six specific regions of target DNA
- Progress through 2 steps by DNA polymerase with strand displacement activity
- Starting structure producing step
- Cycling amplification step
- Large amount of amplicons can be produced by LAMP within 60 minutes which is usually 103-fold or higher as compared to simple PCR
No thermocycler required - isothermal - one temperature required.
Loop-mediated isothermal amplification
28
Q
What is a bacteriophage-based biosensor?
A
- Viruses that invade bacterial cells
29
Q
How do bacteriophage-based biosensors work?
A
- Lytic phages disrupt bacterial metabolism and cause the bacterium to lyse
- Phages co-evolve with their bacterial hosts to recognize and infect their target cells with a high specificity
- The complete infection cycle of a virulent phage usually takes 1-2 h and, by multiplication inside the host cell, offers an inherent amplification step
- Shorten the time for detection
- Detection on the bases of phage-induced lysis
- ATP release
- Other bacterial cytoplasmic markers: activity of bacterial beta-D-galactosidase
- Detection by Phage Amplification Assay
- Genetically modified reporter range
- Using phage or phage components as affinity molecules
