Cannabis TestingiQ-Check Free DNA Removal Solution: A Study to Demonstrate the Impact on Viable
Target Cells

Jennifer Pelowitz, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr, Hercules, CA 94547
Amrita Puri, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr, Hercules, CA 94547
Wendy Lauer, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr, Hercules, CA 94547

Introduction

PCR results in processed foods and environmental samples frequently encounter challenges withfalse positives, primarily arising from the amplification of DNA derived from non-viable cells of targetpathogens. Bio‑Rad developed the iQ‑Check Free DNA Removal Solution (Bio‑Rad Laboratories, Inc.,
catalog #3594970) in 2016 to solve this problem that was observed in environmental samples collectedfrom a dairy product manufacturing plant that was using a phage-based pathogen treatment solution.
The iQ‑Check Free DNA Removal Solution or FDRS, is a non-toxic, enzymatic reagent designed to treat
a variety of food, cannabis, water, and environmental samples to specifically denature extra-cellular
DNA that could result from the application of commonly used antimicrobial treatments in the food andbeverage industry such as heat-processing, dessication, irradiation, disinfectants, etc. The applicationof FDRS in the iQ‑Check real-time PCR kit workflow with cannabis matrices has been AOAC approved
for the detection of Aspergillus, Salmonella, and STEC. This internal study was performed as a proof ofconcept using inoculated environmental sponge samples to determine the impact of FDRS on living cells.
Study Design: Impact of FDRS on viable cells
To challenge the limits of the current validated protocol (10 µl for
15‑30 min), a study was conducted to assess the impact thatFDRS has on viable/live cells (Fig. 1). Environmental sponges
enriched with Listeria Special Broth (LSB, Bio-Rad, catalog#3564703) and Buffered Peptone Water (BPW, Bio-Rad,catalog #12013259) were inoculated with 2.05 x 106 of Listeria
monocytogenes (ATCC 13932) and 3.05 x 106 of Salmonella
Typhimurium (ATCC 14028) respectively. The final concentrationof L. monocytogenes was 3.41 x 104 and 5.08 x 104 for S.
Typhimurium. Analysis was performed by measuring the effectthat various volumes and treatment times of FDRS has on target
microorganisms after enrichment.
FDRS was dispensed in triplicate at volumes of 0, 10, 50, 100 µl
into a deep well microplate. To the 0 µl wells, 100 µl of sterile water
was added to ensure equal dilution volumes was achieved. 100 µlof enriched samples were added to each well and incubatedwithout shaking at 37 ± 2°C for 0, 15, 30, 45, and 60 minutes.
After incubation, samples were extracted using the Easy I or II DNA
extraction protocols and tested using the iQ-Check Salmonella II
PCR and the iQ-Check Listeria spp. kits following the Fast APFs.

0, 10, 50, 100 µl FDRS volumes
0, 15, 30, 45, 60 min
FDRS treatment timeFig. 1. FDRS treatment conditions assessed in the iQ-Check real-time PCR workflow.

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iQ-Check Free DNA Removal Solution: A Study to Demonstrate the Impact on Viable Target Cells

Results
The iQ-Check Listeria spp. Cq values for different volumes ofFDRS added during varied treatment times were similar with littlevariation demonstrating no impact on sensitivity. When the volumeof FDRS was increased over all time points tested, there were onlyminor differences in average Cq value (Fig. 2). At 0 µl FDRS, theaverage difference was 1.86; at 10 µl FDRS, the average difference
was 0.55; at 50 µl FDRS, the average difference was 0.46; at 100 µl
FDRS, the average difference was 0.53. When the incubation
time was increased over all volumes tested, there were only minor
differences in average Cq value (Fig. 3). At 0 min, the average
difference was 1.0; at 15 min, the average difference was 1.44;
at 30 min, the average difference was 2.5; at 45 min, the average
difference was 2.3; at 60 min, the average difference was 2.85. All
positive samples remained positive in all rounds of testing.

Fig. 3. iQ-Check Listeria spp. PCR Curves by Time. FDRS treatment volumes areindicated by the following PCR curves: 0 µl (orange), 10 µl (light blue), 50 µl (green),
100 µl (maroon).

Fig. 2. iQ-Check Listeria spp. PCR Curves by Volume. FDRS treatment timepoints
are indicated by the following PCR curves: 0 min (light blue), 15 min (maroon), 30 min(dark blue), 45 min (purple), 60 min (green).

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iQ-Check Free DNA Removal Solution: A Study to Demonstrate the Impact on Viable Target Cells

For the iQ-Check Salmonella II kit, an impact on the sensitivity
was observed with a 100 µl volume and an incubation time above
45 min. When the volume of FDRS was increased over all time
points tested, only the 100 µl test volume demonstrated significant
differences in average Cq value (Fig. 4). At 0 µl FDRS, the average
difference was 0.57; at 10 µl FDRS, the average difference was0.71; at 50 µl FDRS, the average difference was 1.2; at 100 µlFDRS, the average difference was 3.05. When the incubation time
was increased over all volumes tested, the 45 min and 60 min time
points demonstrated a significant difference in average Cq value(Fig. 5). At 0 min, the average difference was 1.49; at 15 min, theaverage difference was 2.56; at 30 min, the average difference
was 2.95; at 45 min, the average difference was 4.44; at 60 min,the average difference was 4.96. All positive samples remained
positive in all rounds of testing.

Fig. 5. iQ-Check Salmonella II PCR Curves by Time. FDRS treatment volumes areindicated by the following PCR curves: 0 µl (orange), 10 µl (light blue), 50 µl (green),
100 µl (maroon).

Fig. 4. iQ-Check Salmonella II PCR Curves by Volume. FDRS treatment
timepoints are indicated by the following PCR curves: 0 min (light blue), 15 min(maroon), 30 min (dark blue), 45 min (purple), 60 min (green).

Conclusion
This study confirmed that the current AOAC and AFNOR approved
FDRS conditions (10 µl for 15–30 min) do not impact the sensitivityof both iQ-Check kits tested.
The results demonstrate that FDRS used in higher amounts andwith longer incubation times than the standard protocol does
not affect the sensitivity of iQ-Check Listeria spp. method. For
iQ-Check Salmonella II method, an impact on the sensitivity wasobserved with a 100 µl volume and an incubation time above
45 min. From the data generated in this study, Bio-Rad does notrecommend using 100 µl of FDRS for more than 30 min for thedetection of Salmonella environmental sponges.
Visit bio-rad.com/FDRS for more information.
BIO-RAD is a trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. IQ-CHECK
is a trademark of Bio-Rad Europe GmbH in certain jurisdictions. All trademarks used herein
are the property of their respective owner.

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