Food Safety

iQ-Check Free DNA Removal Solution
®

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Increased confidencein results
Seamless integration
with iQ-Check
Real-Time PCR 		
Detection Kits
Easy to use

Since PCR became the method of choice forroutine testing, one of its major challenges has
been the detection of dead cells. With theiQ-Check methods, 104 to 105 dead cells/25 gof matrix would be needed to get a PCR positiveresult. The Free DNA Removal Solution, which
can easily be integrated with iQ-Check DNA
extraction workflows, provides an ideal way toremove free DNA from food and environmental
samples prior to PCR analysis.
While the DNA in intact and living cells isprotected, the free DNA from the sample, andany that is present due to different industrial

Enriched sample
Free DNA

processes, will be degraded. This enzymatic
solution performs as well as ethidium monoazide(EMA) or propidium monoazide (PMA) treatments
but in a much easier and simpler way.
Principle
The degradation of free DNA is performed by aselected enzyme and its specific activation buffer
under optimized conditions. This enzymatic
treatment ensures that the enriched broths aredevoid of free DNA prior to DNA extraction(Figure 1). Then the iQ-Check Lysis Buffer
associated with thermal lysis inactivates theenzyme, allowing for the extraction of DNA fromintact and living cells.

Heat to 37°C for 15–30 min
Live Cells

Free DNA Removal Solution

Sample ready for
DNA extraction with thefree DNA removed

WWW

Fig. 1. Treatment with the Free DNA Removal Solution. Free DNA from dead bacteria or phage-based intervention is present
with live bacteria in the enriched sample. The iQ-Check Free DNA Removal Solution removes the free DNA from the enrichedsample leaving viable bacteria to be detected by PCR. The Free DNA Removal Solution is inactivated during the DNA extraction
step in the iQ-Check method.

The iQ-Check Free DNA Removal Solution is recommended foranalysis of:
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Performance
Treatment with the iQ-Check Free DNA Removal Solution reduces
the signal of dead cells/free DNA by an average of 2 logs or 6 Cqvalues. Contamination with 4 genomes/PCR reaction corresponds
to approximately 4 x 105 dead cells/25 g of matrix. With the FreeDNA Removal Solution, the signal from this level of contamination iscompletely eliminated (Table 1).

Heat processed/cooked matrices (for example, milk powdersand subcomponents)
Products treated with phage application (for example, Listex andSalmonelex)
Environmental samples subjected to cleaning agents andpotentially carrying dead cells

Table 1. Evaluation of the signal detected from serial dilution of dead Listeria
spp. cells tested with the iQ-Check Listeria spp. Real-Time PCR Detection
Kit in the presence and absence of Free DNA Removal Solution treatment.

Using the iQ-Check Free DNA Removal Solution results in anaverage 2–3 log (approximately 6 Cq values) reduction in signal
from free DNA.
ProtocolEnrich the sample according to the appropriate iQ-Check method.
Add 10 µl of activated iQ-Check Free DNA Removal Solution to 100 µlof enriched sample. Incubate 15–30 min at 37°C. Proceed with theiQ-Check method.

Enriched sample

iQ-Check Free DNA
Removal Solution
treatment

Dead cell
contamination,
genomes/PCR reaction

Signal without
iQ-Check Free DNA
Removal Solution,
Cq values

Signal with iQ-Check
Free DNA RemovalSolution, Cq values

400

29.5

Not detected

4031.7

Not detected

435.4

Not detected

An independent evaluation of the iQ-Check Free DNA Removal
Solution was performed on four different matrices using theiQ-Check Listeria spp. Real-Time PCR method in accordance withcurrent AOAC guidelines (Table 2). Using probability of detection(POD) analysis, no statistically significant differences were observed
between the method using the Free DNA Removal Solution and thereference method for all the matrices tested. The iQ-Check Free
DNA Removal Solution does not affect the detection of living cells.

iQ-Check protocol

This protocol has also been successfully tested on milk powders
and subcomponents. Contact your Bio-Rad sales representative formore information.

Table 2. Independent evaluation of the Free DNA Removal Solution using the iQ-Check Listeria spp. Real-Time PCR method in accordance withcurrent AOAC guidelines.
iQ-Check Listeria spp. Kit
MatrixStrainDeli ham

Listeria monocytogenes
ATCC 19115

CheeseStainless steel

Sealed concrete

Listeria welshimeri
ATCC 35897

Listeria innocua
ATCC 33091
and Enterococcus faecalis
ATCC 29212
Listeria monocytogenes
ATCC 7644

MPN, most probable number. CFU, colony forming units.

RESEARCH
INSTITUTE

MPN and CFU/
Test Portion

Number ofSamplesPresumptive
Positive

Confirmed
Positive

Reference
MethodPositive

Equivalency
Criteria

05000

Pass

0.44

20996Pass

3.01

5555Pass

05000

Pass

0.55

20998Pass

3.01

5555Pass

05000

Pass

38 and 420

20556Pass

200 and 2,1005555Pass

05000

Pass

54

20777Pass

170

5555Pass

WORKFLOW COMPARISONThe following diagram compares the iQ-Check Free DNA Removal protocol to current PMA treatment methods. Both are equally efficient,but treatment with the iQ-Check Free DNA Removal Solution is easier and can be carried out in fewer steps.

PMA treatment

iQ-Check Free DNA Removal Solution treatment

Single tube format and micropipet

At least

30 min7 stepsprior toDNA extraction

Deep-well plate format and multichannel micropipet

LABORIOUS
sample handling

3 hr to process
20 samples

5 min

VS2 steps

15–30 minprior toDNA extraction

SIMPLE METHOD

1 hr to process
96 samples

Enriched sample

Centrifuge

Remove supernatant
Add activated Free DNA Removal Solution andenriched sample to deep-well plate
PMA + buffer

Added to the pellet

10 minIncubate at RT in the dark

5 min

10 min

15 to 30 min

Incubate under UV light

Incubate

Vortex and centrifuge

Remove supernatant

orDNA extraction and PCR

DNA extraction with manual or automated iQ-Check protocol

Ordering Information
Catalog # 		 Description

Catalog #3594970

Media for Sample Enrichment
3554179
Buffered Peptone Water Broth, 225 ml x 6 bottles
3564684
Buffered Peptone Water Broth, 500 g
3555797
Fraser ½ Broth, 225 ml x 6 bottles
3553454
Trypto-Casein-Soy (TCS) Broth, 10 ml x 25 tubes
3555703
Listeria Special Broth (LSB), 225 ml x 6 bottles
3564703
Listeria Special Broth (LSB), 500 g3564753
Listeria Special Broth (LSB), 5 kg
3564001
STEC Enrichment Broth, 500 g

iQ-Check Free DNA Removal Solution, 1 vial (175 mg lyophilizedpowder) iQ-Check Free DNA Removal Reagent, 1 tube (9 ml)
iQ-Check 10x Activation Buffer

iQ-Check Real-Time PCR Kits
3578135
iQ-Check Campylobacter Kit, 96 reactions
3578137
iQ-Check Cronobacter Kit, 96 reactions
3578114
iQ-Check E.coli O157:H7 Kit, 96 reactions
3578124
iQ-Check Listeria monocytogenes II Kit, 96 reactions
3578113
iQ-Check Listeria spp. Kit, 96 reactions
3578123
iQ-Check Salmonella II Kit, 96 reactions
3578142
iQ-Check S. Enteritidis Kit, 96 reactions
3578139
iQ-Check STEC VirX Kit, 96 reactions
Coming soon
iQ-Check S. Enterobacteriaceae Kit
Coming soon
iQ-Check S. Typhimurium Kit
Instruments
36000373594911

Industrial Diagnostic CFX96 Touch™ Deep Well Real-TimePCR Detection System, 96 wells (3594990 in Europe)
iQ-Check Prep System

Description

Chromogenic Media for Confirmation
3564748
RAPID'E.coli O157:H7 Dehydrated Base, 100 g
3564744
RAPID'Listeria spp. Dehydrated Base, 500 g
3563694
RAPID'L.mono Agar, 90 mm x 20 plates
3563971
RAPID'Sakazakii Agar, 90 mm x 20 plates
3563963
RAPID'Salmonella Agar, 90 mm x 120 plates

Visit foodscience.bio-rad.com for more information about
Bio-Rad’s food testing products.
Listex and Salmonelex are trademarks of Micreos Food Safety BV.
Bio-Rad’s real-time thermal cyclers are covered by one or more of the following U.S.
patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers:
6,767,512 and 7,074,367.

Bio-RadLaboratories, Inc.
Web site bio-rad.com USA 1 800 424 6723 Australia 61 2 9914 2800 Austria 43 1 877 89 01 177 Belgium 32 (0)3 710 53 00 Brazil 55 11 3065 7550
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France 33 01 47 95 69 65 Germany 49 89 31 884 0 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300
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