Diagnostic Methods & Bioassays Flashcards
What are the 5 components of diagnostic testing operations and bioassays?
- food type and environment
- sample collection plan
- hazard to be tested
- interpretation and decision based on lab results (hazard limits)
- analytical methods used for detection, enumeration, or quantification
What are the possible objects tested on diagnostic testing?
- meat
- whole edible egg after shell removal
- cheese
- whole fish/shellfish after shell, head, tail, and gut removal
- milk
- water
- utensils, equipment, surfaces
What food and environment sampling are done for diagnostic testing?
FOOD: raw ingredient or products in-process, ready for market, at grocery, or ready to eat
ENVIRONMENT: food contact surface, non-food contact surfaces (floors, drains, carts, equipment housing), water, air
What 4 parts are in the sampling plans?
- sample size
- sample type
- weight/volume per sample
- sampling interval per year
What sample size is recommended for diagnostic tests? What interval is usually used? What additional way does the USDA do?
33-60 —> quarterly
50 samples where 5 samples are collected weekly over 10 weeks
How much weight/volume is recommended for sampling different tests?
- meat: 25 g or 100 cm^2 swab
- whole egg, cheese, or fish/shellfish
- milk/water: 100 mL
- utensils, equipment, surfaces: 100 cm^2 swab
What are the 3 types of hazards in foods used for testing?
- Biological: microbiological, natural toxins, parasites
- Chemical: unapproved food or color additives, allergens, pesticide, natural toxins (mycotoxins, shellfish), drug residues
- Physical: metal, glass, sand, sharp materials
How are samples processed for biological hazards?
- mix sample in broth at 1:4 sample to broth ratio
- dilute each sample 10-fold dilution up to 10^-4
- subculture each dilution 8-48 hours in 5 tubes
How are samples processed for chemical hazards?
- collect samples
- grind, chop, homogenize
- inject 10-20 µL sample into analytical equipment
What quantitative test is used for detecting bacteria?
direct bacteria plating
- 1:4 dilution followed by 10-fold dilution up to 10^-4 and culture in agar
- plate 0.1 mL from test tubes and incubate on plates
- culture and count colonies
What are the 2 types of microbes found in the food supply chain?
- indicators or sanitary (GHP monitoring)
- dangerous pathogens (food safety)
What 3 counts are used to measure sanitary procedures? What are they 3 overall indicators for?
- coliform colony count
- mesophilic or aerobic colony count
- E. coli colony count
food quality, shelf-life, GHP at any stage in food supply chains
What are the 11 major dangerous pathogens that are used as indicators of food safety?
- Salmonella - all foods
- Shigella
- Yersinia
- Listeria - almost all foods
- Bacillus cereus - crop foods
- Vibrio spp. - seafood
- STEC - protein-rich foods
- Campylobacter - poultry, milk, water
- Norovirus
- Cyclospora
- Cryptosporidium
(occur in food at low incidence and in few food/animals)
In what 3 ways are samples of microbiological test interpreted?
- 2-class (qualitative/categorical)
- prevalence limit method
- 3-class (numerical/quantitative)
What prevalence demands corrective actions in pig, poultry, and beef? Recall?
PIG - > 6%
POULTRY - > 14%
BEEF - > 4%
(> 0%)
What are decisions based on in two-class decision plans? How are decisions made?
uses qualitative/categorical for microbial concentration in food samples based on presence/absence of pathogens in a food sample
decisions based on 25 g —> no bacteria = satisfactory; bacteria = potentially hazardous
(present/absent, satisfactory/unsatisfactory, accept/reject)
What are decisions based on in three-class decision plans? How are decisions made?
uses numerical values for microbial concentration in food samples based on cfu/g of pathogens
- < 10^2 = satisfactory
- 10^2 - 10^3 = marginal
- 10^3 - 10^4 = unsatisfactory
(satisfactory/borderline/unsatisfactory, accept/doubtful/unsatisfactory)
Pathogen concentrations detected in food:
What is the likely cause of the necessity for proper hygiene action following decisions based on diagnostic results?
inadequate time and temperature control during cooling, storage, processing, or reheating
slow or inadequate cooling, reheating, or cooking of large production volumes
What is the basis for detecting foodborne contaminants?
central dogma - DNA —> RNA —> protein
- DNA-RNA-protein-based test: culture independent diagnostic test (CDIT)
- microbiology and parasitology
- biological activity assays on host cells, tissues, or animals
What are some tests that use DNA, RNA, proteins/metabolites, or nucleic acids/enzymes/antibodies?
DNA - PCR, nucleic acid probe, DNA microarray, whole genome sequencing, metagenomics
RNA - RNA viruses, mRNA, rtPCR
P/M - ELISA, lateral flow assay, gel diffusion assay, fluorescent anitbody, radioimmunoassay, MALDI-TOF, HPLC
NA/E/A - biosensors; bioreceptors = antibodies, enzymes, cells, DNA, phage, molecules; transducers = electrochemical, optical, mass-based
(CDIT)
How are cells and tissues/animals used in bioassays?
CELLS - Chinese hamster ovary cells, Vero cells, mice spleen cells, human peripheral lymphocytes
TISSUES/ANIMALS - mouse lethality test, monkey and kitten emesis test, rabbit and guinea pig skin tests, rabbit and mice ileal loop test
What are the 2 purposes of microbiological tests for food?
- confirms that food is produced in hygienic conditions with effective sanitation detergents/disinfectants, personnel, hygienic status, sanitation programs, HACCP, GHP, and decontamination
- confirms that food is safe by detecting presence of pathogenic microorganisms
What are bioassays? What are they the standard test for? When are results received?
procedure that requires model organisms, tissues, or host cells to test harmful effects of stimuli (chemical, microbe) on living things
- toxicology
- pathogenicity
- dose-response
12-72 hours
What are the 3 objectives of bioassays?
- expose living organisms to different concentrations of potential toxicantd
- observe the effect of the organism’s behavior and survival
- determine if, or at what concentration, a chemical has harmful effects
What are the 3 essential components of bioassays?
- HOST - subject animal, tissue, cells, organells
- AGENT - pathogenic, toxic, drug residue, or sample stimuli
- INTERACTION - response; death, necrosis, degeneration, dysfunction, disease
What are the 3 types of model organisms used for bioassays? What type of study are they used in?
- IN VITRO - cancer, somatic, or immune cells (spleen, WBCs) for cell-based bioassays
- EX VIVO - skin, ileal loop, jejunal loop for tissue-based bioassays
- IN VIVO - mice, guinea pig, rabbit, monkey, dogs, or kittens for animals-based bioassays
What is a dose-response curve? What is the LD50?
relationship between the magnitude of the response of an organism, as a function of exposure (or doses) to a stimulus or stressor (usually a chemical) after a certain exposure time
concentrations where 50% of the population of test subjects dies —> ~43 mg/kg
What is the most powerful toxin?
botulinum toxin —> LD50 = 0.00001 mg/kg
What is the standard test for drug and pesticide residue detection in food?
chromatography (HPLC)
What is the turnaround and sensitivity/specificity of nucelic acid and protein-based culture-independent diagnostic tests?
NUCLEIC ACID (PCR) - results within 1-3 hours, sensitivity of 10^3-10^4 cfu, excellent specificity
PROTEIN - results within 1-3 hours, sensitivity of 10^4-10^5 cfu, good specificity
What are the 4 major nucleic acid-based diagnostic methods of detecting pathogens in food?
- PCR
- multiplex PCR
- rtPCR (quantitative)
- whole genome sequencing (WGS) - becoming more popular
Detection limits of real-time and multiplex PCR:
rtPCR = more sensitive
What are the 2 major immunological methods used to detect antigens or antibodies in food? What do each work best for?
- ELISA - bacterial toxins
- lateral flow assays - pathogen detection
What is required for detection on lateral flow assays?
1000 - 100000 cfu in food samples
(not standardized for wider use yet)
What are the 3 best ways for detecting chemical hazards?
- higher performance liquid chromatography (HPLC) - can detect multiple hazards at once (multi-residue methods)
- ELISA
- bacterial inhibition tests on suspected food samples
How does the USDA FSIS and FDA use bacterial inhibition tests for surveillance?
USDA FSIS - liver, kidney, muscles
FDA - domestic milk, seafood, shelled eggs, honey, feed, plants, and game meat samples
Why does the FDA commonly use chromatography for residue detection?
detects almost all residues in a sample at once —> multiclass multi-residue methods
What are the 8 components assembled in HPLC?
- solvent reservoir - methanol-water, acetonitrile-water
- degasser
- pump
- injector - 1-500 µL test sample
- column filled with silica or polymer beads (normal/polar, reverse/non-polar, ionic, gel, porous)
- UV, IR, fluorescence detectors
- computer software
- waste container
What food material is the FDA responsible for pesticide monitoring?
- plants
- seafood
- domestic milk
- shelled eggs
- honey
- game meat
How does the violation rates for maximum residue limits compare in imported and domestic samples?
violation rate of imported samples is 3-5x higher than domestic samples
What is the bacterial inhibition test commonly used as an inficator for? What 2 samples are most commonly used?
indicators of maximum residue limits of veterinary drugs in carcasses
- kidney
- muscle
What 3 things happen on bacterial inhibition tests if antimicrobials are present in the sample?
- bacteria will not grow
- acid will not be produced
- color will not change (remains purple)
How are microbial inhibition assays performed?
muscle/kidney samples are placed on an already plated agar media and inhibition zones are measured and indicate the presence of drug residues
