Diagnostic Methods & Assays Flashcards
Bioassya and diag methods need 5 components:
- ID your food commodity that you are going to investigate with different diagnostic tests and the env they are in.
- Decide the food commodity, then have a sampling plan.
- Determine hazard type that are targeted: bio, physical, or chemical hazards
- Post lab tests, how do youb interpret the results?
- What are the limits? what is acceptable and rejectable - Analyticla methods used to decide this info
Step 1: Identify the food ________, food _______ or _______ for diagnostic testing
There are a lot of food commodities in the US. Broadly divided into _______ and _______ animal foods, ______ foods. Which ones are you targeting the investigation? is it meat, egg, seafood, milk/milk products OR are you sampling water bodies, air, env in which food is moving? even surfaces, vehicles, humans working with commodity.
Step 1: Identify the food ingredient, food products or
environment for diagnostic testing
There are a lot of food comoddites in the US. Broadly divided into seafood and terrestrial animal foods, plant foods. Which ones are you targeting the investigation? is it meat, egg, seafood, milk/milk products OR are you samplign water bodies, air, env in whihc food is moving? even surfaces, vehicles, humans working with comoddity.
Are you targeting the _________ part of food in process during processing OR targeting _____ product or ______ to eat type of food Or the _________.
Are you targeting the ingredient part of food in process during processing OR targeting end product or ready to eat type of food Or the environment.
Step 2: Describing the _______ plan
Includes:
These include
1. Sample ____
2. sample _____,
3. ______/______ per sample,
4. sampling _______ per year
Step 2: Describing the sample plan
Includes:
These include
1. Sample size
2. sample type,
3. weight/volume per sample,
4. sampling interval per year
FDA and USDA
Sample size they collect most of the time is between ____/____-_____.
Minimum # of sample size that really tells you should not be less than ____. If less than this, results are by _____.
Interval: sample every ________ ( every ____ months) but also sample ______. E.g, USDA samples 5 samples per week over 10 weeks, so in total 50 samples collected. E.g. FDA samples 33 samples per month, so 8 per week. But during an outbreak, you can sample up to 60 because during an outbreak you do not have to wait until 1 week comes.
FDA and USDA
Sample size they collect most of the time is between 30/33-60.
Minimum # of sample size that really tells you should not be less than 30. If less than this, results are by chance.
Interval: sample every quarter ( every 3 months) but also sample weekly. E.g, USDA samples 5 samples per week over 10 weeks, so in total 50 samples collected. E.g. FDA samples 33 samples per month, so 8 per week. But during an outbreak, you can sample up to 60 because during an outbreak you do not have to wait until 1 week comes.
How much weight/volume & what samples to test?
If sampling solid food, like meat = ____ g minimum
eggs, seafood, cheese -> grab ___ of them.
Liquid = _____ mL
if you are just sampling surfaces, such as surface of meats by swabbing or surface of equipment, floor walls, handle, people –> swab around _____ cm squared.
If sampling solid food, like meat = 25 g minimum
eggs, seafood, cheese -> grab 1 of them.
Liquid = 100 mL
if you are juust sampling surfaces, such as surface of meats by swabbing or surface of equipment, flooor walls, handle, people –> swab around 100 cm squared.
Step 3: What is the hazard type I am targeting?
- bio
- viruses, bacteria, protozoa, worms; ~ 31 pathogens within foodborne diseases alone - chem
- food allergens, pesicides, drug residuals, toxins like alagal toxin, myxo toxin, and others - phys
Step 4.1. Processing samples in the Lab
for biological & chemical hazard test
The way you process your sample will differ between biological and chemical hazard analysis. E.g. during bio hazard analysis, you sampled 25 g meat. ____ it down, mix with _____. Broth should be ___x the weight of that meat. How much broth do you have? ____ mL. _______ and _______ wash from the surface of this food. Then you _______ dilute into 5 test tubes. Then you culture 1 mL on _____ plates, then you count the ______ of bacteria per _____ of meat and you convert it to ____. This helps you decide whether this food can enter food ______ _____ or be _____.
E.g. during bio hazard abnalysis, you sampled 25 g meat. Chop it down, nix with broth. Broth should be 4x the weat of that meat. How much broth do you have? 100 mL. Viruses and bacteria wash from the surface of this food. Then you serially dilute into 5 test tubes. Then you culture 1 mL on agar plates, then you count the amt of bacteria per gram of meat and you convert it to kg. This helps you decide whehter this food can enter food supply chain or be rejected.
What are the Two types of microbes in food supply chain?
- Pathogenic ones causing illness for 48 million people annually.
- Non-pathogenic/commensal but still cause food spoilage.
List the Non-pathogenic bacteria that can pose a problem during food processing and explain why they can be a problem.
List the Pathogenic bacteria that can pose a problem during food processing and explain why they can be a problem.
- E.coli
- Coliforms
- Mesophilic or Aerobic colony count
Food is not prepared under hygienic conditions. Shelf life is short, even though they do not cause disease in humans. These tell us whether environment is clean or not, shelf-life, GHP at any stage.
See image
Pathogenic organisms affect certain food commodities.
1. ________ affects everything from vertebrates, seafood, eggs, meat, milk, even plants.
2. _______ is mostly pork
3. _______ mostly animal foods, sprouts
4. __________ mostly poultry products.
5. ________ = seafood
6. ___________ mostly plant foods such as patsta potato rice
7. __________ is also everything
8. ___________ = seafoods
Pathogenic organisms affect certain food commodities.
Salmonella affects everything fromv ertebrates, seafood, eggs, meat, milk, even plants.
Yersinia is mostly pork
E.coli mostly animal foods, sprots
campy mostly poultry products.
vibrio = seafood
bacilusm mostly plant foods such as patsta potato rice
listeria is also everyhting
norovirus = seafoods
Step 4: Interpretation of Lab results for decision
3 options:
PRevalence
decision making where you reject food from the suply chain if the prevalence of this hazard is above a certain set prevalence limit.
e.g. usda limit for slaughter lines –> if amt from 50 samples is above 6% (3/50 = 6%) you have to stop this slaughter line and it needs to be cleaned.
- For poultry it goes up to 14% acceptable limit. (surface of equip)
- sample 25 g of meat, limit is 0. no bacteria shou;d be in food. anything + –> reject b/c it is contaminated
Differnece betwen 2 and 3 class decision
2 = positive negative; contaminated and non contaminated.
- + = reject
- - = pass
3 = rely on counting. you have to count colony forming units. anything < 10^2 is very good b/c less tahn 100.
if count is above 1000 it is dangerous
- if between 100 and 1000 = marginal.
Differnece betwen 2 and 3 class decision
2 = positive negative; contaminated and non contaminated.
- + = reject
- - = pass
3 = rely on counting. you have to count colony forming units. anything < 10^2 is very good b/c less tahn 100.
if count is above 1000 it is dangerous
- if between 100 and 1000 = marginal.
Gram - bacteria –> 2 way decision b/c should be 0
Listeria –> 2 way decision b/c should be 0
Gram + bactria –> 3 way decision
Vibrio should really be less than 3 cfu
Borderline: investigate b/c above acceptable level even though they are not at the level of rejection. There is apthogen build up in suppply chain so need to investigate this. is it the cooking condition? tmperature? cooling system not working properly? washing? where is the problem?
During unsatisfactory condition, must prescribe what? rejection. This is not sufficient alone. You must also write a letter then also do investigton to see whre problem is.
- how do i see hazard
- option1: feed food to expeimental animals maybe mice, rabbit, kitten, puppies, monkeys
- why? if there is a hazard in this suspected food, animals might show vomit, fever, diarrhea, etc. This is called a bioassay.
- option 2: culture for bacteria or you can culture viruses in cell cultures or observe the parasites, cysts.
- option 3: you can observe the DNA or the RNA or the protein of the pathogen.These are the dogma of life. If pathogens here, their DNA and RNA is there so we use this dogma of life. E.g, things that detect their DNA –> what tool can do this? PCR, metagenomics, etc.
RNA, how do you do this?
Toxin, how do you do this? ELISA, flow assay, mulgitoff chromatogaph
Bioassays can be used for the whole organism. if do not have that, what do you use? biopsy e.g. collect from slaughterhouses a skin swab or intestinal segment from slaughterhouses and expose with this toxicant if there is necrosis, dgeneation,m etc.
Microbiological tests for food hygiene & safety programs
We need live animals, expensive.
If can not do this, you can use the biopsy or tissue or intesitne or skin. can also buy cells
expose them with pathogen or food you suspect or drug residuals
Helps us tell i there is a problem.
Can increase dose of hazard
start with 0 mg/kg and go up. The more you increase the concentration of the hazard, the more animals die
50% of herd dies –> LD50
What are The three essential components of bioassays?
in vivo = live animal
ex vivo = outside animal
in vitro = using cell
bacillus causes diarrhea, so give suspected toxin to animal and observe if there are any changes on these cells.
clostridium cases mouse lethality and paralysisl, then death
50% pop will die if we keep increasing the concentration.
LD50 –> 43
Step 5.3. Culture-independent diagnostic test (CIDT)
NA and protien based are very fast, you can get the results within 1-3 hours and their specificity and sensitvity are very good. Do not tell you if orgniasm is really present becase we are looking at the subunit.
HPLC is very poweful toool that FDA is using for residuals in food.
Step 5.3.1. Nucleic acid based diagnostic
methods of pathogens in food
WGS price is becoming cheaper adn cheaper and is almost replacing culture based approach
2 phases
1–> detect like regular PCR
2–> counts pathogen # using DNA
multiplex you can just use several primers together to detect virus, bacteria, protozoa, if hey are present in a single sample.
Direct ELISA –> load sample onto plate and use antibody if + or -
Indirect ELISA –> twoAb are used
Sandwich ELISA –> 3 Ab are used
ELISA is not as easy as PCR
Very effective
Lateral flow is very easy
Put known Ab against Penicillin. This Ab is put on a cellulous membrane with a tunnel around it. Bring sample, load it where it says “s”. Within this tunnel it flows and when it reaches around the test line, if sample is + for penicillin a line will appear. I sample does not have any, it remains white.
Lateral flow is not standardized yet
HPLC can detect up to 800 different substances; drug pesticies, tc.
Can also use ELISA or last option
Chromatography is a widely used method by
FDA to detect residues in food
“River” flowing through machine.
Inject sample by syringe. This liquid that is flowing can be hydrophillics or hydrophobic. Then it goes itnto the column and beads that are oppposite to substance status (hydrophobic/hydrophillic)
Each substance comes at a speocifc time.
Tetrayclcine
Penicillin he said
read all %
if you take milk or meat or kidney of animals, if there is drug in here, bacteria willl die
halo around = drug in it
no halo = no drug in it
