BIOO FOOD AND FEED SAFETY
AuroFlow™
BT Combo Strip Test Kit Manual
Catalog #: 1087-01 & 1087-02
This kit is manufactured to the international quality standard ISO 9001:2008.
ISO CI#: SARA-2009-CA-0114-A
©BIOO Scientific Corp. • 2014
V14.07
TABLE OF CONTENTS
GENERAL INFORMATION ...................................................................... 1
Product Description .................................................................................. 1
Mechanism of Reaction ............................................................................. 1
Sensitivity (Detection Limit) ..................................................................... 2
Kit Contents, Storage and Shelf Life ......................................................... 2
Required Materials Not Provided With the Kit ......................................... 3
Optional Materials Not Provided With the Kit ......................................... 3
BT COMBO STRIP TEST PROTOCOL ................................................... 3
Strip Testing Procedure ............................................................................. 3
Interpretation of Results ........................................................................... 4
Warnings and Precautions ........................................................................ 5
RELATED PRODUCTS .............................................................................. 5
Milk Residue Testing ................................................................................. 5
The AuroFlow™ BT Combo Strip Test Kit is intended for laboratory use only, unless otherwise indicated. This product
is NOT for clinical diagnostic use. AuroFlow is a trademark of Bioo Scientific Corporation (BIOO).
AuroFlow™ BT Combo Strip Test Kit – 1087-01 & 1087-02
GENERAL INFORMATION
GENERAL INFORMATION
Product Description
The AuroFlow™ BT Combo Strip Test Kit is a qualitative and rapid lateral flow assay designed
to detect both beta-lactam and tetracycline antibiotic residues in raw commingled cow’s milk.
This test uses a tetracycline receptor and a novel beta-lactam binding protein which eliminates
the need for a heating block. This test is designed for rapid field use or reference lab settings.
Examples of beta-lactam and tetracycline antibiotics and their limits of detection using the
AuroFlow™ BT Combo Strip Test Kit are shown in Table 1.
The features of the kit are:
 Rapid strip test method – 7 minutes
 Novel binding protein that requires no heating step
 High sensitivity
 High reproducibility
Mechanism of Reaction
The assay uses a competitive colloidal gold based format. The milk sample (200 μL) is added to a
clear plastic reaction vessel, and used to resuspend the lyophilized reagents to a uniform pink color
in the bottom of the microtiter wells. The milk is incubated briefly (3 min) to allow the both antibiotic
binding proteins on the gold particles to engage with any antibiotic present in the milk. The test
strip is then inserted into the sample well with the arrows pointing downward initiating capillary flow
up the strip. Any gold particles that are not complexed with antibiotics present in the milk will bind
to their respective test line, forming a signal (red line) at that position. Each test strip is imprinted
with two test lines and one Control line (C-line). One test line allows for the detection of
tetracyclines, and the other for the detection of beta-lactam antibiotics. If the binding protein on the
gold particle has engaged with either a tetracycline or a beta-lactam antibiotic present in the milk
sample, the gold particle will flow past the test line corresponding to that antibiotic and reach the
C-line. For visual interpretation of the test results, any test line signal intensity that is stronger than
the signal at the C-line indicates a negative result. Signal at any test line which is equal or less
intense compared to the C-line indicates the presence of the corresponding antibiotic in the milk.
The greater the reduction in signal intensity at the test line, the greater the concentration of
tetracycline or beta-lactam antibiotics present in the milk. The assay is also designed to
simultaneously detect tetracycline and beta-lactam antibiotics.
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AuroFlow™ BT Combo Strip Test Kit – 1087-01 & 1087-02
Sensitivity (Detection Limit)
Table 1: Detection limit by visual interpretation method for different beta-lactam and tetracycline
antibiotics by AuroFlow™ BT Combo Strip Test Kit
Beta-lactam Antibiotics
Penicillins
Cefalosporins
Antibiotic (MRL)
Detection
Antibiotic (MRL)
Detection
limit (μg/L)
limit (μg/L)
Penicillin G (4)
3-4 ppb
Ceftiofur (100)
75-100 ppb
Ampicillin (4)
4-5 ppb
Cephapirin (60)
6-15 ppb
Amoxicillin (4)
4-5 ppb
Cefazolin (50)
35-50 ppb
Cloxacillin (30)
4-8 ppb
Cefoperazone (50)
5-20 ppb
Oxacillin (30)
4-8 ppb
Cephquinome (20)
10-20 ppb
Dicloxacillin (30)
5-8 ppb
Cephalonium (10)
4-8 ppb
Nafcillin (30)
15-30 ppb
Cefacetrile (125)
30-50 ppb
Cephalexin (100)
250-300 ppb
Cefuroxime (n/a)
150-200 ppb
Tetracycline Antibiotics
Antibiotic (MRL)
Tetracycline (100)
Chlorotetracycline (100)
Doxycycline (0)
Oxytetracycline (100)
Detection
limit (μg/L)
50-100 ppb
15-50 ppb
5-20 ppb
50-70 ppb
Kit Contents, Storage and Shelf Life
The AuroFlow™ BT Combo Strip Test Kit has the capacity for 96 (Cat # 1087-01) or 192 (Cat #
1087-02) determinations. The shelf life is 24 months when the kit is properly stored.
The positive and negative control vials contain lyophilized raw milk samples, and must be
reconstituted with 1 mL of distilled water before use. The positive control vial is spiked with 4
ppb Penicillin G and 100 ppb of Oxytetracycline. The reconstituted raw milk controls are stable
for 48 hours at 4°C. Additional positive and negative control vials are available separately (Cat
# 108710).
Cat# 1087-01
Kit Contents
Reaction wells
Test strips
Positive control
Negative control
Pipette tips
Amount
4 x 24 (96 total)
4 x 24 strips (96 total)
1 vial
1 vial
100 tips
Storage
4°C
4°C
4°C
4°C
RT
Amount
8 x 24 (192 total)
8 x 24 strips (192 total)
2 vials
2 vials
200 tips
Storage
4°C
4°C
4°C
4°C
RT
Cat# 1087-02
Kit Contents
Reaction wells
Test strips
Positive control
Negative control
Pipette tips
A 96-well plastic frame is also provided in each kit to anchor the reaction vessels in place
during testing, and can be re-used as required.
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AuroFlow™ BT Combo Strip Test Kit – 1087-01 & 1087-02
Required Materials Not Provided With the Kit
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Timer/Watch
Additional positive and negative controls (Cat # 108710)
Polypropylene tubes, clean vials, or small glass test tubes
Distilled water
Scissors to cut strip caps
Optional Materials Not Provided With the Kit

QuickSTAR Strip Reader (Cat # 1952-02)
Note: this product was developed using the QuickSTAR strip reader, and is highly recommended
for optimal performance. For customers already possessing the QuickSTAR strip reader, or wishing
to use an alternate reader, please contact Bioo Scientific at [email protected] for
instructions on how to program the reader for use with this kit.
BT COMBO STRIP
TESTSTRIP
PROTOCOL
BT COMBO
TEST PROTOCOL
Strip Testing Procedure
1.
2.
3.
4.
5.
6.
7.
8.
Using scissors, carefully cut the number of reaction wells and remove test strips
appropriate for the number of samples that will be tested and allow reagents to reach
ambient temperature (25˚C +/- 5). Ensure that unused wells remain firmly capped.
Promptly re-store the remaining components at 4˚C. To maintain assay timing do not run
more than 6 tests at the same time.
Shake the milk vigorously, aliquot sample into a small polypropylene tube or clean vial
(1.5 mL – 15 mL), and allow milk sample to reach ambient temperature (25˚C +/- 5). For
faster milk temperature equilibration, pipette 1 mL of cold milk into a small glass test tube.
The milk should reach the minimal 20˚C in less than 5 min.
Attach a disposable pipet tip to the end of the pipettor (Cat # 106711).
Insert the pipet tip into the milk sample, depress the plunger of the pipettor to the first stop,
then slowly release the plunger to aspirate 200 μL of milk into the pipet tip.
Position the pipet tip (loaded with milk) over a reaction well and depress the plunger to
completely expel the milk sample into the well. When more than one sample is being
analyzed, note the order in which they are placed in the wells.
Using the same pipet tip, aspirate the sample up and down about 10 times to completely
resuspend the lyophilized reaction particles in the milk, while avoiding bubbles. The
sample should turn a uniform pink color. After resuspending the particles, remove and
discard the pipet tip.
Incubate the sample for 3 min at ambient temp (25˚C +/- 5). During this time, label one or
more test strips as needed for the number of samples being tested.
After 3 min, insert the bottom of the test strip into the well containing the milk sample (see
Figure 1). The strip should be inserted such that the arrows are pointing down. Be sure
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AuroFlow™ BT Combo Strip Test Kit – 1087-01 & 1087-02
the strip is oriented vertically (not leaning to the side) and is inserted all the way to the
bottom of the well. Set a timer for 4 min.
9. After 4 min, remove the strip and place it on a horizontal surface with the unmarked side
facing up.
10. Visually examine the intensity of the signal at each test line and compare the intensity to
the C-line. Interpretation of results: Signal at a test line which is more intense than the
signal at the C-line indicates the milk sample is negative for presence of that category of
antibiotics. Signal at a test line which is equal to or less intense than the signal at the
C-line indicates the presence of that respective category of antibiotics in the milk sample.
See Figure 1 for representative results.
Note: It is recommended to have the same incubation time for each sample. If more than
one sample is being tested, offset the timing for each reaction by 1 minute to ensure that
the test strip is inserted exactly 3 minutes after incubation period and 4 minutes after
insertion into the well. For most accurate interpretation, the assay results must be read
within 5 min of removing the test strip from the well.
Interpretation of Results
Figure 1. Test Strip Orientation and Test Interpretation
The following diagram if for visual interpretation only. If using the QuickSTAR strip reader refer to
reader instructions provided with the unit. If the β-lactam or Tetracycline lines are equal or less
intense than the C-line, then the milk sample is positive for the respective antibiotic class. If both
test lines are less intense than the C-line, the milk sample is positive for both β-lactams and
Tetracyclines.
Orientation
Interpretation
Negative
β-lactam
Positive
Tetracycline β-lactam &
Positive Tetracycline
Positive
C-line
β-lactam
Tetracycline
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AuroFlow™ BT Combo Strip Test Kit – 1087-01 & 1087-02
Invalid results
The test is invalid if no colored line appears in the C-line area even if a colored line appears in
the test line area. Re-test milk sample if broken lines are observed. Colored lines which
appear after 20 minutes are not diagnostic and should be ignored. If the sample does not flow
up the dipstick, the milk sample may be clotted, invalidating the test result. To avoid milk
sample clotting, always use fresh milk.
Warnings and Precautions
Bioo Scientific recommends that you read the following warnings and precautions.
Periodically, optimizations and revisions are made to the kit and manual. Therefore, it is
important to follow the protocol included with the kit. If you need further assistance, you may
contact your local distributor or Bioo Scientific at [email protected]
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Do not use the kit past the expiration date.
Do not re-use pipette tips or polypropylene tubes.
Be sure unused wells remain tightly sealed with the plug caps.
Incubations of assay should be timed as precisely as possible.
Always store test strips in provided vials to maintain stability.
Positive samples should always be re-tested alongside a positive and negative control.
Material safety data sheets (MSDS) are available for this kit.
Quality control (QC) reports are available for this kit.
BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its
products are made are of standard quality. There is no warranty of merchantability of this product, or of the
fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or
consequential damage, or expense arising directly or indirectly from the use of this product.
RELATED PRODUCTS
RELATED PRODUCTS
Milk Residue Testing
Product
Catalog Number
AuroFlow™ Beta-lactam Strip Test Kit (96 reactions)
AuroFlow™ BTS Combo Strip Test Kit (96 reactions)
AuroFlow™ Sulfonamide Strip Test Kit (96 reactions)
AuroFlow™ Melamine Strip Test Kit (96 reactions)
AuroFlow™ Fluoroquinolone Strip Test Kit (96 reactions)
QuickSTAR Strip Reader
1067-01
1116-01
1111-01
1091-01
1102-01
1952-02
Bioo Scientific Corporation
7050 Burleson Rd.
Austin, TX 78744 USA
Tel: 1.888.208.2246
Fax: (512) 707-8122
Made in USA
BIOO Food & Feed Safety Products
[email protected]
[email protected]
www.biooscientific.com
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