Charm Sciences, Inc.

659 Andover Street, Lawrence, Massachusetts, 01843-1032, USA

Tel: +1.978.687.9200 | Fax: +1.978.687.9216 | Email: info@charm.com | www.charm.com
Copyright 2009 Charm Sciences Inc. ROSA (Rapid One Step Assay) is a registered trademark of Charm Sciences, Inc. Protected
under one or more of the following patents: U.S. 6,475,805; 5,985,675; EP 1044360
.
OM-400-002 26-Mar-2009 Page 1 of 4
Operators Manual: Ethanol Extraction
ROSA

Aflatoxin (Quantitative) Test

Quantitative Lateral Flow Test for Aflatoxin in
Grains and Feeds
Introduction
The ROSA Aflatoxin (Quantitative) test is a lateral flow immunoassay. Aflatoxin is extracted from a sample
using 50% ethanol. The extracted sample interacts with colored beads and the intensity of color in the test
zone is read as parts per billion (ppb) aflatoxin by the ROSA-M Reader.
Sensitivity: 0 to 100 ppb Aflatoxin
Limit of Detection (LOD): < 2 ppb
Commodities Verified: Barley, Brewers Rice, Corn, Corn Gluten Feed, Cottonseed, Cottonseed Hulls,
Cottonseed Meal, DDGS, Milled Rice, Oats, Peanut Hulls, Rice Bran (Hi-fat), Rice Fractions, Rice Hulls,
Sorghum, Wheat, Wheat Midds
Order Codes and Kit Contents
Reagents and
Supplies to run
Aflatoxin:
Reagents and
Supplies to run
DON:
Reagents and
Supplies to run
Fumonisin:
Reagents and
Supplies to run
Ochratoxin:
Reagents and
Supplies to run
Zearalenone:
LF-MYCO-AFQ-100K 100 Tests
LF-MYCO-DONQ-100K 100 Tests
LF-MYCO-FUMQ-100K 100 Tests
LF-MYCO-OCHRA-G-100K 100 Tests
LF-MYCO-ZEARQ-100K 100 Tests
LF-MYCO-AD-200K 100 Tests 100 Tests
LF-MYCO-AFD-300K 100 Tests 100 Tests 100 Tests
LF-MYCO-AFDO-400K 100 Tests 100 Tests 100 Tests 100 Tests
LF-MYCO-AFDOZ-500K 100 Tests 100 Tests 100 Tests 100 Tests 100 Tests
List of materials to perform test
A list of equipment, required materials, and order codes are supplied in the Material Addendum: Materials
and Supplies for ROSA Mycotoxin Test Kits sent with each kit.
Equipment and Reagent Preparation
Prepare 50% ethanol by mixing 500 ml ethanol and 500 ml deionized or distilled water. Mix well,
clearly label, and store at room temperature in a tightly sealed container.
Place clean incubator on level surface. Incubator temperature should be at 451C (strip indicator
should be green at 45).
Inspect test strips to ensure that packaging is not damaged. Dented sample compartments should be
re-shaped to allow strips to easily fit into incubator.
Predispense 1.0 ml AFQ Dilution Buffer to a micro-centrifuge tube for each sample to be tested.
Handling, Disposal, and Decontamination of Aflatoxin Samples
Refer to local or regional recommended procedures for the handling, disposal, and decontamination of
aflatoxin samples.
OM-400-002 26-Mar-2009 Page 2 of 4
Reagents and Storage
Test Strips Store refrigerated at 0 to 7C in tightly-closed, supplied container.
To open foiled container, remove and save plastic lid with foil lined foam insert to
reseal container. Lift foil tab and peel foil seal off container. Discard foil seal.
In high humidity, limit condensation by opening container after it is warmed to
room temperature (20 to 30 minutes from the time the container is removed from
refrigerator).
Inspect desiccant indicator. Beads inside desiccant packets should be blue. Discard
test strips if desiccant beads turn white or pink.
Remove from container the number of test strips to be used. Keep these test strips
at room temperature for up to 12 hours and discard any unused test strips.
Immediately reseal container using lid with foil lined foam insert and return to
refrigerated storage.
Negative
Control
Negative Control is prepared with 100 l (0.100 ml) 50% ethanol added to 1.0 ml
AFQ Dilution Buffer.
To run Negative Control, use 300 l in step 4 of Procedure.
Positive
Control
Aflatoxin B1 Positive Control is supplied dry. Store at 0 to 7C.
Reconstitute Aflatoxin B1 Positive Control with 500 l (0.500 ml) 50% ethanol
followed by 5.0 ml AFQ Dilution Buffer and mix for 30 seconds. Allow to stand for 10
minutes at room temperature. Mix again before use. Rehydrated Aflatoxin B1
Positive Control is equivalent to Diluted Extract prepared from 20 ppb Aflatoxin B1
in corn.
Store rehydrated Aflatoxin B1 Positive Control at 0 to 7C for up to 1 week or
aliquot and freeze at -15C or below for up to 2 months. Store thawed control at 0
to 7C and use within 24 hours of thawing.
To run Aflatoxin B1 Positive Control, use 300 l in step 4 of Procedure.
AFQ Dilution
Buffer
Use at room temperature (18 to 30C).
Micro-centrifuge tubes can be predispensed with 1.0 ml AFQ Dilution Buffer.
Store AFQ Dilution Buffer bottle and any unused predispensed tubes at 0 to 7C.
Daily Calibration Strip Check
Enter performance mode in reader by pressing ESC and then 5. Press ENTER to activate Calibration
Strip mode.
LOWCAL will appear on the reader sample line. Insert the low calibration strip into the reader and
press ENTER. Strip must test in specified ranges listed on back of calibration strip.
HIGHCAL will then appear on the reader sample line. Insert the high calibration strip into the reader
and press ENTER. Strip must test in specified ranges listed on back of calibration strip.
Press ESC after counting both Low and High Calibration Strips to reset reader.
If Calibration Strips do not perform in specified ranges, discontinue use and contact Charm Sciences
for assistance.
Weekly Negative and Positive Control Check
Test Negative Control and Positive Control to verify performance of equipment and test strips. Read
the Negative and Positive Control test strips on the MYCO Channel (3-line mode) using MATRIX 03.
Valid Control READING ranges are:
o Negative Control: 3 ppb
o Positive Control: 10 to 30 ppb
If Controls do not perform in specified ranges, discontinue use and contact Charm Sciences for
assistance.
OM-400-002 26-Mar-2009 Page 3 of 4
Extraction and Sample Preparation
1. Obtain a representative sample and mill/grind to a 20-mesh particle size. Mix the sample.
2. Weigh 10 g
*
ground sample and add to a clean extraction container.
*
Charm Sciences Inc. recommends 50 g samples.
3. Add 30 ml 50% ethanol to the sample.
NOTE: For Cottonseed Hulls, Peanut Hulls, Rice Hulls and Wheat Midds, add 50 ml 50% ethanol.
4. Blend or shake for 3 minutes. Allow sample to settle (1 minute) to obtain sample extract.
5. If particles are present after settling, centrifuge to clarify sample extract.
To centrifuge, transfer sample extract (1.0 - 1.5 ml using transfer pipet) to a labeled micro-
centrifuge tube and centrifuge for 10-30 seconds to obtain clarified extract.
6. Prepare additional sample extracts (up to 4 for quad incubator) following steps 1-5.
7. Pipet 100 l sample extract to a predispensed (1.0 ml AFQ Dilution Buffer) micro-centrifuge
tube, cap, mix, and label. This is the Diluted Extract. Repeat for additional samples.
NOTE: for Wheat and Wheat Midds filter each Diluted Extract by drawing into 1 ml syringe and
passing sample through Minisart RC15 syringe filter. This filtered sample is the Diluted Extract to be
used for analysis.
Procedure
1. Label test strip to identify sample.
2. Open incubator lid and place test strip in ROSA-M Incubator with
flat side facing up.
3. While holding strip flat on incubator, use tab to peel tape back to
indicated line to expose sample compartment. Avoid bending the
white wick and sponge under tape.
4. Slowly pipet 300 l (15 l) Diluted Extract or Control into
side of sample compartment at incubator indicator line.
5. Reseal tape over sample pad compartment by pressing. When
testing multiple samples, complete the peel, pipet, and reseal
steps on each strip before going on to the next strip.
Note: Add Diluted Extracts or Controls to all strips within 1
minute. Quad incubator can incubate 4 samples simultaneously.
6. Close lid on incubator, and latch. Solid red timer light will
automatically start when the lid is closed.
7. Incubate for 10 minutes. A 2-minute beeper and a test
complete light (yellow blinking) will indicate the end of incubation.
8. Remove test strip(s) and read on ROSA-M Reader. Read
within 2 minutes of incubation completion. After strip removal,
lower but do not latch the incubator lid.
Avoid
lifting
wick!
OM-400-002 26-Mar-2009 Page 4 of 4
Visual Inspection
Hold sample pad in the down position to inspect test strip. Do not squeeze sample pad. Wipe foreign
matter (dust, etc.) off test strip.
The test is INVALID if any of the following are observed:
C (Control) line is missing, smeared, or uneven.
Either T1 or T2 (Test) lines are uneven.
Diluted Extract or Control is obscuring the T1, T2, or C lines.
Beads do not flow past T1, T2, or C lines.
If test is INVALID re-test the Diluted Extract or Control.
DO NOT PUT INVALID STRIPS IN ROSA-M READER.
Reader Interpretation
ROSA-M Reader results are quantitative parts per billion readings. They are automatically stored in
memory, and may be recalled or downloaded to a printer or computer.
Insert clean and valid test strip into ROSA-M Reader. Slide into slot,
with sample compartment in the up position, until it stops. Read results
on MYCO Channel (3-Line Mode
1
) with appropriate MATRIX
2
. If
desired, enter SAMPLE and/or OPERATOR. Press ENTER to read.
READING: The number displayed is the concentration of aflatoxin (ppb)
in the sample.
Note 1: ROSA-M Reader Manual instructs how to toggle between 2-line and 3-line modes.
Note 2: The appropriate MATRIX numbers are as follows:
MATRIX 03: Barley, Brewers Rice, Corn, Corn Gluten Feed, Cottonseed, Cottonseed
Meal, Distillers Dried Grains with Solubles, Milled Rice, Oats, Rice Bran
(Hifat), Rice Fractions, Sorghum. Wheat.
MATRIX 05: Cottonseed Hulls, Peanut Hulls, Rice Hulls, Wheat Midds.
Warranty Information
Charm Sciences, Inc. (Charm) warrants this product to be free from defects in materials and workmanship, when stored under appropriate conditions and given normal, proper
and intended usage, until the expiration of its stated shelf life, or, if none is stated, for one year from the date of delivery of this product to the end-user purchaser. THIS
WARRANTY IS IN LIEU OF ALL OTHER WARRANTIES, EXCEPT TITLE, WHETHER STATUTORY, EXPRESS, IMPLIED (INCLUDING WARRANTIES FOR
MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE AND ALL WARRANTIES ARISING FROM COURSE OF DEALING OR USAGE OF TRADE). The warranty
provided herein and the data, specifications and descriptions of Charm products appearing in Charms product literature may not be altered except by express written agreement
signed by an officer of Charm. Representations, oral or written, which are inconsistent with this warranty or such product literature are not authorized and if given, should not be
relied upon.
In the event of a breach of the foregoing warranty, Charms sole obligation shall be to repair or replace, at its option, any product or part thereof that proves defective in materials
or workmanship within the warranty period, provided the customer notifies Charm promptly of any such defect. The exclusive remedy provided herein shall not be deemed to have
failed of its essential purpose so long as Charm is willing to repair or replace any nonconforming Charm product or part. Charm shall not be liable for consequential, incidental,
special or any other indirect damages resulting from economic loss or property damages sustained by any customer from the use of its products.
MYCO
DATE 03/25/2009
SAMPLE 1
MATRIX 00
READING 0005
ppb
RESULT