Quiz 3 Content Flashcards
Why detect foodborne pathogens? [2]
- 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
Describe traditional culture-dependent pathogen detection.
- 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
Describe the isolation of E.coli O157:H7 using traditional culture-dependent detection.
-
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
How is Tellurite Cefixime - Sorbitol MacConkey Agar used to isolate E. coli O157:H7? [3]
- Cifixime inhibits other enteric bacteria
- Potassium tellurite inhibits non-O157 STEC
- The sorbitol containing medium differentiates nonsorbitol fermenters from sorbitol fermenters
Describe the isolation of Salmonella using traditional culture-dependent methods.
-
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
What biochemical analysis generally follows the traditional culture based method?
- Serotyping (based on the slide agglutination test)
What is serotyping?
- 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
What is the O antigen?
Outermost portion of the lipopolysaccharide
Somatic antigens
What is the H antigen?
Flagellar protein
Phase 1/Phase 2 flagella
What is the slide agglutination test?
- Antigens are assayed with antibodies on an inert surface
Describe the isolation of Campylobacter.
-
Campylobacter blood free selective agar (CCDA)
- Charcoal Cefoperazone Deoxycholate
- Defoperazone and Amphotericin B were used as supplements for selective growth of Campylobacter
Describe the isolation of Listeria monocytogenes.
- 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
What are the advantages of the traditional culture-dependent isolation methods? [2]
- Sensitive (can detect pathogens even in trace amounts)
- Reliable (still represent the gold standard)
What are the disadvantages of the traditional culture-dependent isolation methods? [2]
- Time consuming (at least 3 days required for results)
- Labour intensive
Why are rapid detection methods needed? [3]
- Detection results are desired within a shorter period of time
- Some foods are perishable
- Require special storage conditions
Describe the workflow of rapid detection methods.
- Dilute and enrich for 10-28 hours
- Prepare sample
- Conduct assay
What are the targets of current rapid detection technologies?
- ELFA & bacteriophage-based biosensor target surface molecules
- PCR and other molecular detection systems target DNA and ribosomes (inside the cell)
Describe the history of detection of Salmonella and L. monocytogenes.
How does ELISA work?
- 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
Describe ELFA.
- 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
What are the steps in PCR?
- Denaturing (95°C)
- Annealing (40-60°C)
- Extension (72°C)
Describe real-time PCR: The Taqman Probe Method
- 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
Why isn’t a detection phase necessary in Real-time PCR?
- Signal is measured and analyzed at the end of each cycle during amplifcation
What are the advantages of PCR? [4]
- Adaptable to new targets (can always design new primers and probes)
- Potential for high specificity
- Good multiplexing (multiple primers may be designed)
- Rapid & sensitive
What are the disadvantages of PCR? [2]
- Likely to detect dead organisms
- Some assays are relatively labour intensive
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.
Loop-mediated isothermal amplification
What are the advantages of next-gen isothermal amplification? [2]
- Doesn’t need a thermo-cycler
- Cost effective
Next-generation method
What is a disadvantage of isothermal amplification?
Not good for multiplexing
This is because designing primers is much more complicated than in PCR.
What is a bacteriophage-based biosensor?
- Viruses that invade bacterial cells
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
TC-SMAC agar is used for the detection of […].
TC-SMAC agar is used for the detection of E. coli.
[…] utilizes a substrate that yields a fluorescent for foodborne pathogen detection.
ELFA utilizes a substrate that yields a fluorescent for foodborne pathogen detection.
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.
What do product-line specific criteria apply to?
Applies to all test kits within the product line.
List the pathogen specific criteria. [9]
- Inclusivity
- Exclusivity
- Diagnostic sensitivity
- Diagnostic specificity
- Analytical sensitivity
- Reproducibility
- Repeatability
- Compatability
- Validation status
What is inclusivity?
Ability to detect different strains and/or subtypes of the target organism
Pathogen-specific criteria
What is exclusivity?
Ability to yield negative results with non-target organisms.
Pathogen-specific criteria
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.
Pathogen-specific criteria
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
Pathogen-specific criteria
What is analytical sensitivity?
Detection limit: before enrichment (CFU/25 g) or after enrichment (CFU/mL)
Pathogen-specific criteria
What is reproducibility?
Ability to perform reproducibly in different laboratories and with different personnel
Pathogen-specific criteria
What is repeatability?
Ability to produce the same results in the same laboratory with the same equipment and personnel
Pathogen-specific criteria
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?
Pathogen-specific criteria
What is validation status?
Certified by ISO, AFNOR, AOAC International, and/or Health Canada
Pathogen-specific criteria
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
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
What is differentiation?
- Should not detect nonviable organisms.
Product line-specific criteria
What is ruggedness?
Should not be affected by small changes in protocol or small deviation from recommended environmental conditions for equipment.
Product line-specific criteria
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
Product line-specific criteria
What is control of cross-contamination?
- Should have multiple barriers to prevent cross contamination
Product line-specific criteria
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
Product line-specific criteria
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
Product line-specific criteria
What is speed with regard to product specific criteria?
Significantly reduced time relative to standard method
Product line-specific criteria
What is throughput?
Number of samples allowed, time to run the assay, batch processing availability
Diagnostic specificity is the probability that a test will correctly classify a negative test as negative.
True or False?
True
Avoid false positive results
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.
Avoid false positive results
Diagnostic specificity refers to avoiding false positive results.
True or False?
True.
Diagnostic specificity is the probability that a test will correctly classify a negative test as negative and avoid false positive results.
Diagnostic specificity refers to avoiding false negative results.
True or False?
False.
Diagnostic specificity is the probability that a test will correctly classify a negative test as negative (and avoid false positive results).
Diagnostic sensitivity is the probability that a test will correctly classify a positive test sample as positive.
True or False?
True.
Avoid false negatives
Diagnostic sensitivity is the probability that a test will correctly classify a negative test sample as negative.
True or False?
False.
Diagnostic sensitivity is the probability that a test will correctly classify a positive test sample as positive (and avoid false negatives).
Diagnostic sensitivity refers to avoiding false negative results.
True or False?
True.
Diagnostic sensitivity is the probability that a test will correctly classify a positive test sample as positive, and avoid false negative results.
Diagnostic sensitivity refers to avoiding false positive results.
True or False?
False.
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).
