iQ-Check Free DNA Removal Solution for Food, Water, and Environmental Samples

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Since PCR became the method of choice for routine testing, one of its major challenges has been the potential detection of dead cells leading to unconfirmed positive results. Bio-Rad developed the Free DNA Removal Solution (FDRS) which can easily be integrated with current iQ-Check PCR extraction workflows. It provides an ideal way to remove free DNA from food, water, and environmental samples prior to PCR analysis with a non-toxic, easy to use procedure. While the DNA in intact and living cells is protected, the free DNA from the sample, and any that is present due to different industrial processes, will be degraded.

Free DNA Solution

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 enriched sample 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.

Solution for Enriched Food and Environmental Samples

The degradation of free DNA is performed by a selected enzyme and its specific buffer under optimized conditions. This enzymatic treatment degrades free DNA in the enriched sample prior to DNA extraction. The iQ-Check Lysis Buffer then inactivates the enzyme, allowing for the extraction of DNA from intact and living cells. This non-toxic enzymatic solution performs as well as ethidium monoazide (EMA) or propidium monoazide (PMA) treatments but in a much easier and simpler way.

Enriched Food and Environmental Samples


The iQ-Check Free DNA Removal Solution is recommended for analysis of:

  • Heat processed/cooked matrices (for example, milk powders, and subcomponents)
  • Products treated with phage interventions
  • Environmental samples submitted to cleaning agents and potentially carrying dead cells Using the iQ-Check FDRS protocol results in an average 2–3 log (approximately 6 quantification cycle [Cq] values) reduction in signal from free DNA. The protocol is also adaptable to the iQ-Check Prep Automation System

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An independent evaluation of FDRS was performed on four different matrices using the iQ-Check Listeria spp. real-time PCR method in accordance with current AOAC guidelines (Table 1). Using probability of detection (POD) analysis, no statistically significant differences were observed between the method using FDRS and the reference method for all the matrices tested. FDRS does not affect the detection of living cells. This protocol has also been successfully tested on milk chocolate, raw milk cheese, chicken carcass, stainless steel.


Table 1. Independent evaluation of the Free DNA Removal Solution using the iQ-Check Listeria spp. Real-Time PCR method in accordance with current AOAC guidelines.

iQ-Check Listeria spp. Kit
Matrix
Strain
MPN and CFU/
Test Portion
Number of Samples
Presumptive Positive
Confirmed Positive
Reference Method Positive
Equivalency Criteria
Deli Ham Listeria monocytogenes
ATCC 19115
0
0.44
3.01
5
20
5
0
9
5
0
9
5
0
6
5
Pass
Pass
Pass
Cheese Listeria Welshimeria 
ATCC 35897
0
0.55
3.01
5
20
5
0
9
5
0
9
5
0
8
5
Pass
Pass
Pass
Stainless Steel Listeria innocua
ATCC 33091
and Enterococcus faecalis
ATCC 29212
0
38 and 420
200 and 2,100
5
20
5
0
5
5
0
5
5
0
6
5
Pass
Pass
Pass
Sealed Concrete Listeria monocytogenes
ATCC 7644
0
54
170
5
20
5
0
7
5
0
7
5
0
7
5
Pass
Pass
Pass

In the scope of the AFNOR validation renewal and extension, the FDRS demonstrated a real efficiency to reduce the false positive rate from 13.3% down to 0% when using the Listeria spp. Fast PCR run in environmental samples.

AOAC and AFNOR reports are available on request.

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Validation: Within the scope of international validation authorities, the performance of our methods are verified by third party laboratories following strict guidelines demonstrating their optimal accuracy and quality.

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Solution for Legionella Testing

FDRS is recommended for analysis of environmental water samples for Legionella testing. Using FDRS results in up to 2.5 log (approximately 6 Cq values) reduction in signal from free DNA when introduced into the Aquadien DNA Extraction Kit Protocol prior to real-time PCR detection with iQ-Check.

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Aquadien DNA Extraction Kit

An evaluation of FDRS was performed on water from six clean and dirty environmental samples using the iQ-Check Quanti Legionella spp. and L. pneumophila real-time PCR kits. In five samples, free DNA was removed with FDRS treatment. Two samples achieved equivalency between PCR and culture methods. Differences between PCR and culture results were due to viable but non-culturable (VBNC) bacteria that were not detectable in culture. Data representing the differences between sample results are compared in table 2.


Table 2. FDRS action on environmental water samples and comparison between qPCR and culture results on Legionella spp. and L. pneumophila targets.

Targets
qPCR without FDRS, log GU/L
qPCR with FDRS, log GU/L
FDRS Action, log
Culture, log
CFU/L
Δ log Culture vs. qPCR with
FDSRS
FDRS Action and Observation
Interpretation
Legionella spp.
7.5
6.5
–1.0
4.7
–1.8
Free DNA removed; high presence of VBNC
VBNC not detected by culture
Legionella spp.
7.1
5.6
–1.5
4.7
–0.9
Free DNA removed; medium presence of VBNC
VBNC not detected by culture
Legionella spp.
7.6
5.2
–2.4
4.7
–0.5
Free DNA removed; low presence of VBNC
VBNC not detected by culture
L. pneumophilia
6.1
5.4
–0.7
5.5
0.1
Free DNA removed
PCR and culture are equivalent
L. pneumophilia
4.4
3.7
–0.7
3.9
0.2
Free DNA removed
PCR and culture are equivalent
L. pneumophilia
5.2
5.0
–0.2
3.7
–1.4
No free DNA; no presence of VBNC
VBNC not detected by culture
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Resources

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iQ-Check FDRS Protocol

Learn how the Free DNA Removal Solution works to help eliminate free DNA from food, water, and environmental samples with a simple protocol.

 

Documents