Detection Of Food Bourne Illness Flashcards
What is the main advantage of ATP-bioluminescence?
Portable, low cost, rapid estimation of residual bacterial contamination
What is a disadvantage of ATP-bioluminescence?
Does not indicate the type of bacteria detected
What does Polymerase Chain Reaction (PCR) amplify?
Specific ID genes/portions in the genome of target microorganisms
What are the three steps of Polymerase Chain Reaction (PCR)?
- Denaturation
- Annealing
- Elongation
What temperature is used for the denaturation step in PCR?
95°C
What is the purpose of the annealing step in PCR?
Primers bind to the target DNA
What enzyme is used during the elongation step of PCR?
Taq polymerase
What is the limit of detection for conventional PCR?
10² - 10³ cells/gr tested food
What is the main difference between simplex PCR and multiplex PCR?
Simplex PCR detects one target sequence, while multiplex PCR detects multiple target sequences
What are the advantages of multiplex PCR?
- Detection of several different pathogenic bacteria
- Improved identification of closely related pathogenic bacteria
What is the principle of Real-Time Quantitative PCR (qPCR)?
Specific detection and amplification of specific genes with online acquisition of results
What type of method is Reverse Passive Latex Agglutination (RPLA)?
Immunological method used for detecting bacterial toxins in food
What does ELISA stand for?
Enzyme-linked immunosorbent assay
What is a key feature of ELISA?
Detection based on colorimetric reaction rather than visible clumping
What is the limit of detection for ELISA in terms of bacteria?
10³ - 10⁴ CFU/ml
What is the primary function of reverse transcription in RT-PCR?
Conversion of RNA into complementary DNA (cDNA)
What are some foodborne pathogens that are RNA viruses?
- Human Norovirus
- Hepatitis A virus
- Hepatitis E virus
Fill in the blank: PCR requires two _______ molecules.
single strand (SS)
True or False: Multiplex PCR is less sensitive than conventional PCR.
True
What is the incubation time typically required for RPLA?
< 60 minutes
What is the role of antibodies in ELISA?
Bind to the target antigen to form antigen-antibody complexes
What type of result does ATP-bioluminescence provide?
Semi-enumeration of bacteria
What is the temperature range for the annealing step in PCR?
50-62°C
What are the components of the PCR reaction mixture?
- DNA extracted from the sample
- Two primers
- dNTPs
- Taq polymerase
What is the detection method used in qPCR?
Fluorescence emitted by newly synthesized DNA copies
What are the disadvantages of traditional culture methods?
- Enrichment may be required
- Results take several days
- Viable, infectious organisms may not be detected
What is the primary target for immuno-based detection methods?
Specific biomarkers/proteins
What is the primary advantage of ELISA over agglutination methods?
More sensitive and permits direct detection of toxins and bacteria
What is the role of Taq polymerase in Real time quantitative PCR (qPCR)?
Assemble the DNA components in the two ss DNA molecules
What happens during the denaturation/melting phase of qPCR?
Heating samples at 95ºC to separate DNA into single strands
What is the purpose of fluorescent components in qPCR?
Bind to the melted DNA and emit fluorescence to be measured
During which phase do primers bind to single-stranded DNA in qPCR?
Annealing
What does the release of dyes during elongation in qPCR indicate?
Emission of fluorescence proportional to DNA copies synthesized
How is the enumeration of microorganisms calculated in qPCR?
Based on the number of PCR cycles required to produce detectable fluorescence
What is the limit of detection (LOD) in qPCR for microorganisms?
~100 microorganisms
What are some advantages of Real time quantitative PCR (qPCR)?
- Quicker results (≤ 3 hours) * Accurate enumeration of microorganisms * More sensitive than conventional PCR
What are some disadvantages of qPCR compared to conventional PCR?
- More expensive * Requires specialized equipment * Needs trained personnel
What is the total time required for conventional food microbiology methods?
Up to 6-7 days
What are some selective and differential media used for detecting Bacillus cereus?
- Polymyxin * Egg Yolk Mannitol Bromothymol Blue (PEMBA)
What are good practices for food safety in the food chain?
- Good Operating Practices * Good Manufacturing Practices * HACCP
What percentage of food safety issues are caused by bacteria?
> 60%
What is the main focus of modern food microbiology?
Detection of foodborne microorganisms
What technology measures ATP for rapid detection of bacterial contamination?
ATP-luminescence technology
What is the significance of minimally processed food (MPF) in modern food microbiology?
MPF are perishable with a shelf life generally ≤ 10 days
What is the selective agent in Brilliance Listeria agar?
Lithium chloride, polymyxin B, and nalidixic acid
What does MacConkey agar differentiate based on?
Lactose fermentation
What is the role of bile salts in Deoxycholate agar?
Prevent growth of non-enteric bacteria
What are conventional/traditional methods used to detect living bacteria in a sample matrices
•Based on culture
•Enumeration of colonies formed on
solid media (agar plates)
Disadvantages of traditional methods
Problems with plate cultures
•Enrichment may be required
•Numbers are often underestimated
•Sometimes viable, infectious organisms not detected.
Timescale
•Repeated incubations mean that results take several days
Name modern detection methods
Two main categories:
1 - Nucleic acid-based methods - PCR
2 - Immuno - based methods - Reverse Passive Latex agglutination and ELISA
