Quiz 3 Content Flashcards
(62 cards)
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
24
What are the disadvantages of PCR? [2]
* Likely to detect dead organisms
* Some assays are relatively labour intensive
25
What is LAMP?
* 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.
## Footnote
Loop-mediated isothermal amplification
26
What are the advantages of next-gen isothermal amplification? [2]
* Doesn't need a thermo-cycler
* Cost effective
## Footnote
Next-generation method
27
What is a disadvantage of isothermal amplification?
Not good for multiplexing
## Footnote
This is because designing primers is much more complicated than in PCR.
28
What is a bacteriophage-based biosensor?
* Viruses that invade bacterial cells
29
How do bacteriophage-based biosensors work?
* 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
30
TC-SMAC agar is used for the detection of [...].
TC-SMAC agar is used for the detection of ***E. coli***.
31
[...] utilizes a substrate that yields a fluorescent for foodborne pathogen detection.
**ELFA** utilizes a substrate that yields a fluorescent for foodborne pathogen detection.
32
What is the purpose of pathogen-specific criteria?
Evaluate assays for various pathogens (e.g., *Salmonella* and *L. monocytogenes*) even when they are part of the same product line.
33
What do product-line specific criteria apply to?
Applies to all test kits within the product line.
34
List the pathogen specific criteria. [9]
* Inclusivity
* Exclusivity
* Diagnostic sensitivity
* Diagnostic specificity
* Analytical sensitivity
* Reproducibility
* Repeatability
* Compatability
* Validation status
35
What is inclusivity?
Ability to detect different strains and/or subtypes of the target organism
## Footnote
Pathogen-specific criteria
36
What is exclusivity?
Ability to yield negative results with non-target organisms.
## Footnote
Pathogen-specific criteria
37
What is diagnostic sensitivity?
The probability that a test will correctly classify a positive test sample as positive (avoid false negative results).
| Overlap between this criteria and exclusivity.
## Footnote
Pathogen-specific criteria
38
What is diagnostic specificity?
The probability that a test will correctly classify a negative test sample as negative (avoid false positive results).
| Otherwise known as limit of detection
## Footnote
Pathogen-specific criteria
39
What is analytical sensitivity?
Detection limit: before enrichment (CFU/25 g) or after enrichment (CFU/mL)
## Footnote
Pathogen-specific criteria
40
What is reproducibility?
Ability to perform reproducibly in different laboratories and with different personnel
## Footnote
Pathogen-specific criteria
41
What is repeatability?
Ability to produce the same results in the same laboratory with the same equipment and personnel
## Footnote
Pathogen-specific criteria
42
What is compatibility with regards to pathogen-specific criteria?
* Compatability with existing and standard enrichment media
* Are specific enrichment media or proprietary nonstandard media required?
## Footnote
Pathogen-specific criteria
43
What is validation status?
Certified by ISO, AFNOR, AOAC International, and/or Health Canada
## Footnote
Pathogen-specific criteria
44
Differentiate between diagnostic sensitivity and diagnostic specificity.
**Sensitivity**: probability that a test will correctly classify a positive test sample
**Specificity**: probability that a test will correctly classify a negative test sample
45
List the product line specific criteria. [12]
* Differentiate viable and nonviable organisms
* Ruggedness
* Internal positive controls
* Control of cross contamination
* Different target pathogens
* Compatibility with food matrix
* Speed
* Throughput
* Equipment size
* Ease of use
* Cost
* Product support
46
What is differentiation?
* Should not detect nonviable organisms.
## Footnote
Product line-specific criteria
47
What is ruggedness?
Should not be affected by small changes in protocol or small deviation from recommended environmental conditions for equipment.
## Footnote
Product line-specific criteria
48
What are internal positive controls?
PCR-based methods can yield false-negative results, so an internal positive control can provide assurance that a negative test result is a true negative and not a false negative due to inhibition
## Footnote
Product line-specific criteria
49
What is control of cross-contamination?
* Should have multiple barriers to prevent cross contamination
## Footnote
Product line-specific criteria
50
Why have different target pathogens in a product line?
Should have assays available for all key foodborne pathogens and target organisms of concern to specific end user
## Footnote
Product line-specific criteria
51
What is compatibility with respect to product-line specific criteria?
Some food matrices can cause high numbers of false-negative results or inhibit the detection assay
## Footnote
Product line-specific criteria
52
What is speed with regard to product specific criteria?
Significantly reduced time relative to standard method
## Footnote
Product line-specific criteria
53
What is throughput?
Number of samples allowed, time to run the assay, batch processing availability
54
Diagnostic specificity is the probability that a test will correctly classify a negative test as negative.
True or False?
True
## Footnote
Avoid false positive results
55
Diagnostic specificity is the probability that a test will correctly classify a positive test as positive.
True or False?
False.
Diagnostic specificity is the probability that a test will correctly classify a negative test as negative.
## Footnote
Avoid false positive results
56
Diagnostic specificity refers to avoiding false positive results.
True or False?
True.
## Footnote
Diagnostic specificity is the probability that a test will correctly classify a negative test as negative and avoid false positive results.
57
Diagnostic specificity refers to avoiding false negative results.
True or False?
False.
## Footnote
Diagnostic specificity is the probability that a test will correctly classify a negative test as negative (and avoid false positive results).
58
Diagnostic sensitivity is the probability that a test will correctly classify a positive test sample as positive.
True or False?
True.
## Footnote
Avoid false negatives
59
Diagnostic sensitivity is the probability that a test will correctly classify a negative test sample as negative.
True or False?
False.
## Footnote
Diagnostic sensitivity is the probability that a test will correctly classify a positive test sample as positive (and avoid false negatives).
60
Diagnostic sensitivity refers to avoiding false negative results.
True or False?
True.
## Footnote
Diagnostic sensitivity is the probability that a test will correctly classify a positive test sample as positive, and avoid false negative results.
61
Diagnostic sensitivity refers to avoiding false positive results.
True or False?
False.
## Footnote
Diagnostic sensitivity is the probability that a test will correctly classify a positive test sample as positive, and avoid false negative results.
So, to clarify:
Sensitivity: Ability to avoid false negatives (correctly identify individuals with the condition).
Specificity: Ability to avoid false positives (correctly identify individuals without the condition).
