Dr. Erin Bowers, Professor at Iowa State University and Pat Frasco Director of Sales Milling & Grain for Neogen presented mycotoxin testing best practices

1. Mycotoxin Testing
a hands-on workshop
Erin Bowers, PhD
January 8, 2019

2. Mycotoxins are not entirely avoidable,
even with GAP and GMP in place.
• Fungal by-products that affect
grains and other products
globally
• We don’t grow crops in a
sterile environment
• Risk prediction for strategic
testing is possible
Image ©Gary Munkvold
Climate
Host
Mycotoxins
Pathogen

3. There are five principal mycotoxins of
concern in grains
Mycotoxin Fungus Cereal
crops
affected
Favorable conditions
Aflatoxin Aspergillus flavus,
Aspergillus parasiticus
Corn
Rice
Hot, drought
Deoxynivalenol Fusarium graminearum,
Fusarium culmorum
Corn
Wheat
Barley
Cool, wet
Fumonisin Fusarium verticillioides,
Fusarium proliferatum
Corn Warm-hot, drought during
grain maturity
Zearalenone Fusarium graminearum,
Fusarium culmorum
Corn
Wheat
Cool, wet
Ochratoxins Penicillium verrucosum Corn
Wheat
Barley
Oats
Warm-hot, humid
conditions

4. Harvest 2019

5. DON is Iowa’s main issue (and we aren’t the
only ones)

6. Advisory Levels for Deoxynivalenol
in Livestock Feed
Class of Animals
Feed Ingredients & portion of the
diet
DON level in
ingredients and
(finished feed)
Ruminating beef and feedlot
cattle older than 4 months
10 ppm (10 ppm)
Ruminating dairy cattle older
than 4 months
Grain and grain by-products not to
exceed 50% of the diet
10 ppm (5 ppm)
Chickens
Grain and grain by-products not to
exceed 50% of the diet
10 ppm (5 ppm)
Swine
Grain and grain by-products not to
exceed 20% of the diet
5 ppm (1 ppm)
All other animals
Grain and grain by-products not to
exceed 40% of the diet
5 ppm (2 ppm)

7. The end user determines the level of
“acceptable” mycotoxins

8. Why test?
• Inform marketing decisions
• Meet contract demands
• Safety assurance
• FSMA-control the hazard to minimize
risk (and subsequent liability)

9. FDA’s action, advisory, and guidance
levels for aflatoxins, deoxynivalenol, and
fumonisins
• Aflatoxin 20-300 ppb depending on ingredient/animal
• 20 ppb general commerce
• 0.5 ppb aflatoxin M1 in milk
• Deoxynivalenol 5-10 ppm in ingredients, 1-10 ppm in
finished feed depending on animal
• 1 ppm in finished wheat products for human use
• Fumonisins 5-100 ppm in ingredients, 1-50 ppm in
finished feed depending on animal
• 2-4 ppm for human food, depending on product
Testing for other mycotoxins may be appropriate,
depending on market

10. There is high variability in mycotoxin
levels among individual kernels of grain
• 207,000 ppb aflatoxin in individual corn kernel (62.1µg
aflatoxin in a 0.3g kernel)
• 8 of these in a bushel of corn puts it at the 20 ppb limit for use in general
commerce Shotwell, O., Goulden, M. and Hesseltine, C. 1974. Aflatoxin:
distribution in contaminated corn. Cereal Chem 51:492-499.
Johansson, A.S. et. al., C. 2000. Testing Shelled Corn for Aflatoxin, Part II: Modeling
the Observed Distribution of Aflatoxin Test Results. JAOAC Intl. 83:1270-1278.
~90,000 kernels in a bushel of corn = appx. 70-80 contaminated kernels @ 20 ppb

11. Mycotoxin contaminated grains are
not distributed uniformly in grain lots
This makes representative sampling difficult
Hypothetical distribution of an incoming load of corn
with an average aflatoxin level of 10 parts-per-billion
(ppb)
0 ppb 0 ppb 0 ppb 0 ppb 0 ppb
0 ppb 0 ppb 0 ppb 0 ppb
100
ppb

12. Practical Implications
Obtaining a representative sample means you
have to obtain a random sample and sample
enough units to have a chance of obtaining an
accurate representation of whatever you are trying
to measure.

13. Sampling matters! It
contributes the most
variability to a mycotoxin
test result, followed by
sample preparation and
then analysis.
3 steps in a mycotoxin testing procedure
and 3 sources of variance in a mycotoxin
test result
Johansson, A.S. et. al., C. 2000. Testing Shelled Corn for Aflatoxin,
Part I: Estimation of Variance Components. JAOAC Intl. 83:1264-
1269.
• Sampling-when, how (procedure), how many
units/increments
• Sample preparation-processing and selecting test
portion
• Analysis-extraction and quantification

14. Getting your sample
 Sample representativeness is increased by
collecting incremental samples and combining
them together together make a composite sample
 The laboratory sample is taken from the composite
sample (this is what gets ground up…hint,
hint...this is bigger that what is used for your test!)
 Ground up laboratory sample is mixed and then
sub-sampled to obtain the test portion

15. FGIS laboratory sample sizes for official tests
FGIS Mycotoxin Handbook, Ch. 4

16. FGIS has prescribed sampling methods
for various grain transportation units
• This is a time-consuming practice
• May not be practical or feasible to use for every load at some
grain facilities
• Prescribed for compliance sampling (aflatoxin)
• Just remember, for your own business purposes, to
define your lots in logical, defensible ways.
Sampling patterns for flat-bottom trucks or trailers
containing grain less than 4-feet deep (top) and
more than 4-feet deep (bottom)
United States Department of Agriculture Federal Grain Inspection Service. 1995. Grain
Inspection Handbook - Book 1 Grain Sampling. United States Department of Agriculture
Grain Inspection, Packers & Stockyards Administration.

17. Once you have your representative sample,
grind the whole sample and divide it to obtain
the test portion
• Want small particle size (most recommend at
least 75% of material pass through 20-mesh
sieve; GIPSA 95%)
https://www.seedburo.com/po
p.asp_Q_poptype_E_2_A_ima
geName_E_3010x.jpg
https://www.seattlecoffeegear.co
m/bunn-bulk-commercial-coffee-
grinder

18. We are here for a mycotoxin testing
workshop…
Prior to analysis, I hope you have put a great
deal of thought into proper sampling and
sample preparation.

19. Analysis
• Ground test sample → extract → dilute → analyze
• Many options, best choice depends on your
situation
• Many considerations with +/- for each
• In-house/external lab
• Analysis method
• HPLC
• Lateral flow
• ELISA

20. Rapid test kit-applicability
• Water vs. solvent based
• Matrix appropriateness
• Personnel training
• Skills, accuracy, consistency
• FGIS “approved” mycotoxin rapid test kits

21. Feed & Grain Live
Reveal Q+ MAX
for Aflatoxin
Raptor Integrated Platform
Product Training

22. Reveal Q+ MAX for Aflatoxin
Product Specifications:
Product code: 8088
Limit of detection: 2 ppb
Range of quantitation: 3-50 ppb
With dilution: 3-300 ppb
Test per kit: 25 samples
Testing time: 6 minutes
Shelf Life: 12 months
Validated commodities:
Barley, Black beans, Brown rice, Carioca beans, Cashews,
Corn, Corn Meal, Corn silage, Cottonseed, DDG syrup,
Grass silage, Oat flour, Pistachio, Popcorn, Rice, Sorghum,
Wheat, Walnuts, Almond, Peanuts, Peanut butter
Note: some commodities may need to be pH adjusted or run at
extraction ratios outside the standard 1:5 ratio.
Reveal Q+ MAX for Aflatoxin is a
single step lateral flow assay based
on a competitive immunoassay
format. The kit is designed to detect
the four principle types of aflatoxin:
B1,B2, G1, and G2 with superior cross
reactivity. The kit utilizes an aqueous
extraction eliminating the need for
harsh solvents in your lab.

23. Why MAX?
• Aqueous extraction – no need for harsh solvents
• Common MAX 1 extraction – able to use one sample and test
for several mycotoxins
• Better cross reactivity – more accurate recovery on all aflatoxin
analytes (B1,B2,G1,G2)
• AOAC certification
• FGIS certification

24. Equipment Required
Extraction Supplies:
• Collection cups with lids
• Collection tubes with caps
• Filter syringes, Whatman #4 filters,
or centrifuge and mini-centrifuge
tubes
• Distilled or deionized water
• MAX 1 packets
Equipment:
• Agri-Grind or equivalent
• Scale
• Timer
• Graduated cylinder
• Rock-it Shaker or a hand
• Sample rack
• 250 µL and 400 µL pipettor and tips
• Raptor System

25. Kit Contents
Materials provided:
• 25 Reveal Q+ MAX for
Aflatoxin test strips
• 25 red sample dilution cups
• 25 clear sample cups
• 1 bottle sample diluent
• 25 MAX 1 Aqueous Extraction
packets
• Kit instructions

26. Sample preparation and extraction
1. Obtain a representative
sample (min 100 grams).
Grind and weigh out a 10 g
sample.
2. Pour entire contents of 1- MAX 1 Aqueous
Extraction Packet into the container cup.

27. 3. Add 50 mL distilled or
deionized water to
the sample cup.
4. Shake sample vigorously for 3 minutes
by hand or use mechanical shaker
(Rock-It shaker)
Sample preparation and extraction

28. 5. Allow sample to
settle
Filter sample using a
Whatman #4 filter
paper.
OR
Sample preparation and extraction
6. Filter sample using
a filter syringe.

29. You may also pipette 1 mL of sample into a 2.0 mL
micro-centrifuge tube, and centrifuge for 30 seconds
Sample preparation and extraction

30. Test Procedure
1. Add 250 µl of sample diluent in a
red sample dilution cup.
2. Add 250 µl of sample extract.
Mix by pipetting up and down
5 times.

31. Test Procedure
3. Insert test strip into the
Raptor cartridge
4. Insert cartridge into
any of the three
Raptor ports
5. Scan lot information if
needed from the QR
code located on the
strip tube

32. Test Procedure
6. Add 400 µl of sample to
the back of the Raptor
Cartridge
7. System will start
automatically or you can
press the Next button. You
can now start an additional
sample in the other two
ports.
8. Results will be
displayed at the
conclusion of the 6
minute incubation

33. Dilution Procedure
For Samples greater than 50 ppb you must dilute and re-test
1. Add 100 µl original sample filtrate to a sample collection tube
2. Add 500 µl distilled or deionized water. Mix well
3. Add 250 µl sample diluent (pink bottle) to a red sample dilution cup
4. Add 250 µl diluted sample (from step 2) to the red sample dilution cup with sample diluent
5. Mix by pipetting up and down 5 times
6. Insert test strip into the Raptor cartridge
7. Place cartridge in any of the three Raptor ports
8. Add 400 µl of sample to the back of the Raptor Cartridge
9. System will start automatically or you can press the Next button
10. Results will be displayed at the conclusion of the 6 minute incubation
11. Remember to multiple the result by 6.

34. Available Training Resources
YouTube video
Procedure Page
Web-cam training

35. For more information, contact your Neogen Representative
(800) 234-5333
www.neogen.com
Ordering Information:
8088 – Reveal Q+ MAX for Aflatoxin
(25 samples- includes extraction packets)
9680 – Raptor Integrated Analysis Platform
9681 – Raptor Cartridges

36. Questions?
Thanks for your interest in
mycotoxin testing solutions