A novel enzymatic treatment to remove contaminating free DNA in bacteriophage-treated samples
for use in routine testing
Laurent Jain, André Quintanar, Jérôme Benoist, Sophie Henaux, Fanny Margotteau, Florence Préaudat, Aurore Compoint, Jean-Philippe Tourniaire, Sophie Pierre, Jean-François Mouscadet
Bio-Rad Laboratories, Food Science Division, Marnes La Coquette, France
Results

Introduction
A common hurdle of PCR based assays for detection of microbial pathogens
in food is often amplification of DNA from dead cells. In routine testing, thefeasibility of confirming presumptive positive results is a key performanceindicator for a laboratory or a method. With regard to molecular methods, sucha capability might be thwarted by the presence of free bacterial DNA originating
in food processes that aim at sterilizing food products (e.g. thermal, chemicalor irradiation processes). Food processing itself may be an intrinsic source of
free DNA contamination. This is the case when bacteriophage spraying is used
as an intervention. For instance Listex™, which aims at preventing Listeria, may
contaminate samples with DNA originating in the phage purification process from
Listeria cell culture. To date, only tedious protocols involving hazardous chemicals
with Ethidium Monoazide (EMA) or Propidium Monoazide (PMA) treatments areavailable to get rid of such free DNA.
The aim of this study was to evaluate an enzymatic approach to suppress free
Listeria DNA present in the food sample due to Listex treatment without affecting
the PCR outcome from living cells.

Methods

Table 1. Efficiency of an enzymatic pretreatment to remove contaminating DNA from Listex solution		
Without treatment
		
Listeria
Internal
PCR target
spp.
ControlA
Listeria
spp.

Pure
18.48
N/A*
25.44
10-1
20.64
33.64
27.42
10-2
23.74
31.82
29.93
10 fold
10-3
27.12
30.86
32.87
serial dilutionof Listex
10-4
29.67
31.43
34.13
10-5
32.76
31.94
38.04
10-6
37.10
31.90
N/A
10-7
39.52
32.28
N/A
* IC was not amplified due to competition with strong positive signal

Pretreated with enzymatic mix
BCInternal
Listeria
Internal
Listeria Internal
Control
spp.
Control
spp.
Control
N/A*
31.32
31.45
32.14
32.08
32.20
32.10
32.21

25.74
27.59
30.84
33.57
36.70
39.98
N/A
N/A

33.66
31.11
31.69
32.20
32.24
32.03
32.05
32.08

26.48
28.36
31.32
34.29
36.57
40.22
N/A
N/A

33.50
31.31
31.77
32.04
31.94
32.00
31.97
32.02

We studied the interference of the FDRS with the detection of Listeria in a food sample. As shown in Table 2, enrichment of 25g portions of cheese that were initially spiked with 36 CFU of Listeria yielded a positive PCR witha Cq equal to ≈24. Serial dilutions of the enrichment in the absence of Listex indicated a dilution limit equal to 10-4, as determined by the highest dilution at which 100% of the 10 samples were positive. Treating the samples
with the activated Free DNA Removal Solution did not modify this dilution limit demonstrating that the pretreatment did not affect the detection of living cells.
In contrast, when Listex was added to the dilutions, all samples including the 107-fold dilution gave rise to a PCR positive signal due to the contaminating DNA. A Cq value of ≈37was observed on the enrichment in
absence of living bacteria (0 CFU) corresponding to the DNA load brought by Listex. A constant Cq value of ≈37 was observed for dilutions 10-5 to 10-7, demonstrating that below the 10-4 dilution limit of the Listeria target,
contaminating free DNA was the main contributor to the positive signal. Implementation of the enzymatic pretreatment rescued the PCR signal specifically due to living cells, as demonstrated by the recovery of dilution limit
comparable to the one obtained in absence of Listex contamination. Again, the IC signal was never affected, thus reinforcing the conclusion that the enzyme pretreatment was inactivated during the extraction step andtherefore did not impact the PCR amplification of cell DNA. It is also important to note that a 5-6 Cq decrease was observed for the Listex solution in absence of Listeria inoculum, indicating that efficiency of the pretreatment
did not vary with the presence of the cheese matrix.
Table 2. Free DNA cleaning from L. monocytogenes culture contaminated with Listex 		Presence of Listeria DNA in Listex

Serial dilutions of Listex were added to Listeria Special Broth (LSB) and incubated 24 hr at 30°C. Then
3 different enzymatic mixes were added to 100µL aliquots in the presence of a specific activating buffer.
These enzymatic mixes (named free DNA removal solution or FDRS A, B & C) were designed to be
specifically inactivated during the iQ-Check® DNA extraction protocol at high temperature (Figure 1). Aftera short incubation at 37°C, the DNA was extracted from these aliquots and amplified using the iQ-Check
Listeria spp. assay according to the kit specifications. Cq values for Listeria targets and Internal Control (IC)™
obtained in the presence or absence of the enzyme mix were compared.

Quick Guide

It was previously suggested that treating cheese with Listex may lead to Listeria PCR positive results.
Therefore, our first goal was to demonstrate the presence of free Listeria DNA in the Listex solution, which
may result in unconfirmed positive PCR result for treated samples. As shown in Table 1, Listex solution gave
rise to positive Listeria DNA PCR, even on 10 7 fold dilutions (Cq = 39.52). Enzymatic pretreatment resulted ina significant Cq increase (Mean increase value: A: 5.9 Cq, B: 7Cq, C: 7.47 Cq), demonstrating that the Listex
solution contained Listeria DNA that was reduced by an approximate 2 Log factor upon treatment with the
enzyme mixes. Moreover, the IC Cqs were constant regardless of the protocol, thereby indicating that the
enzymatic pretreatment did not affect the amplification of DNA present in the sample following the lysis/
extraction step. Altogether these results show that Listex contains free Listeria DNA that can be removed byan enzymatic pretreatment of samples prior to iQ-Check Listeria PCR amplification.

Without Listex

Unspiked
Spike level before enrichment

357-8135 • iQ-Check Campylobacter0
36 CFU/23gr
10-1
10-2
10-3
10-4
PERFORMANCE
10-5 TESTED
10-6
RESEARCH INSTITUTE
10-7™ Salmonella II357-8123• iQ-Check
357-8135•
iQ-Check
Campylobacter
10-fold dilutionsof enrichement
Easy I Extraction Deepwell
ProtocolAOAC Extraction II Deepwell Protocol
Quick
Guide Standard

Listeria monocytogenes detection in cheese in the presence of Listex™

PERFORMANCE TESTED

Quick Guide

In the presence of Listex 				Not activated FDRS B
Activated FDRS B
Not activated FDRS B
Activated FDRS B
			Mean of Listeria spp. 		
Mean of Listeria spp.		
Mean of Listeria spp.		
Mean of Listeria spp.
		% of positive results Cq values (10 replicates)% of positive results Cq values (10 replicates)% of positive results Cq values (10 replicates)% of positive results Cq values (10 replicates)
0%
100%

N/A
23.46

0%
100%

N/A
23.15

100%
100%

37.10
24.20

20%
100%

42.02
23.66

100%
100%
100%
100%
30%
0%
0%

26.46
29.82
32.83
36.20
39.04
N/A
N/A

100%
100%
100%
100%
70%
20%
0%

26.32
29.40
32.30
36.10
39.53
38.69
N/A

100%
100%
100%
100%
100%
100%
100%

27.14
30.21
33.47
35.74
36.96
37.13
37.36

100%
100%
100%
100%
60%
20%
20%

26.66
29.81
33.16
36.34
39.84
40.98
42.14

L. monocytogenes (ATCC 35152) was spiked at 36 CFU/25g portions of cheese in 225ml
of LSB.
Inoculum Deepwell ™
Easy
I Extraction
AOAC
357-8135• iQ-CheckProtocol
Campylobacter
Raw
Meat Samples
Food,
Carcass
RinseorSpongeSamples
was confirmed by plating on TCS medium. After 24 hr at 30°C, enrichment was serially diluted in a control
Food,toCarcass
or Sponge
Samples
Guide
Easy
IRinseExtraction
DeepwellProtocol
AOAC
broth supplemented
with and without Listex. The 10-fold dilution series wasQuickthen submittedthefree DNA
4 hrs•
Enrichthesampleinpre-warmed
buffered
peptone water (25 g in
Food
Food,
Carcass Rinse or Sponge Samples
removalsolution: 100µl was incubated with reagent at 37± 42°C
real-time
hrs for 30 min. DNA extraction and 225
37°Cml), 10 hrs ± 2 hrs at 37°C
10 hrsFood
37°C Cq± 2 hrs
PCR analysis
were performed with•theiQ-Check
Listeria
spp.
valuesfor1 4ml
Listeria
andICtargets
were+
Enrichthe sample
in supplemented
Bolton
Broth
(25
g
in
225
ml),
hrs
• Collectof decantedFinally,
enrichedwesampleand place
it in a well ofona Listex treated cheese. In order to optimize the treatment, we compared two FDRS formulations (Table 3). For all dilutions, the mean Cq value for Listeria was ≈32 on cheese treated Listex
testedFood1 ml
Suppl.
+• limit
Enrichofthe
sampleinfromsupplemented
Bolton Broth(25 FDRS
g in 225directly
ml),
37°C
4 hrsmethod
at 37°C was calculated
20 hrs The sensitivity
deepwellplate
75determined.
of
the
whole
and
expressedasthedetection
Suppl.
BoltonsamplesBolton
subjected+• Enrich the sample in supplemented
Broth (25tog ininactive
225 ml), enzymatic mix B. Unspiked samples or 10 -fold dilutions gave rise to PCR positive signal due to the high content of free DNA from Listex (equivalent to 10 bacterial genomes per gram of
4 hrs at 37°C
20 hrs37°C
41°C
Suppl.
•
Seal
the
plate
with
a
plastic
sealingtape
Bolton• Transfer to 41°C for 20 hrs BPW
Broth
10 replicatesof each dilution.
4 hrs at 37°C
20 hrs41°C
cheese). Pretreatment with activated enzymatic mix B allowed to get rid of the Listex free DNA without affecting the detection of the DNA from living bacteria. Mix B activated at 37 ± 2°C for 15 min was sufficient to recover
BoltonRESEARCH INSTITUTEPERFORMANCE TESTEDRESEARCH INSTITUTE• Transfer(Shaketo 41°Cfor 20 hrs
stomacher bag to homogenize, and allow to decant before

Broth
41°C

• Transfer to 41°C for 20 hrs

SpongeCqCarcasssimilartoone
observed in untreated samples. In contrast, the enzymatic mix D failed to efficiently remove free DNA with 50% of the uncontaminated samples being still positive due to the presence of free DNA
Free DNA cleaning from Listex — Carcass
treated Rinse
cheese inoculated with Carcass
L. monocytogenes
Carcasscollecting.
Rinse
SpongeAvoid
collectinga large
fragments
ofthe
food
debris)Carcass
Rinse
Carcass
Sponge•Rinsecarcass
in
400
ml
BPW
•
Spongethecarcass
Listex.
The FRDS method was performed on Listex treated 25g cheese portions inoculated with
6.4carcass
CFU
of• Rinsecarcassin 400 mlfrom
BPW• Sponge• Spongecarcass• Rinsein L.
400
ml BPWthe thecarcassBrothfor 1 minforml20for 1 minthe deepwell plate at 2,250
20 min• Add
ml 2x BF-BEB• Addg25
2x min
BF-BEB
for above
1 min• Centrifuge
monocytogenes.
After the enrichment step, two versions of the
enzymemix25
andD) were24(Bhrs as shown
24 hrs• Add
25 ml 2x
BF-BEB
Table
3.
Comparisonof enzymatic mixes on Listex-treated cheese inoculated with L. monocytogenes
2,250
g
24
hrs
• Remove
30 ml andadd•
Carefully
removethesealingtape*•
Incubate24hrs
at 42°C• Remove 30 ml and add• Incubate
24 hrs •atRemove
42°C of30themlandadded to the samples and incubated at 37 ± 2°C for 15 min on a 10 foldserialdilutionseriesto samples.
30 mladd
2x BF-BEB• Incubate
24shakinghrs at and
42°C
without
under
42°C
•
Place
the
deepwell
plate
in
the
DW
40
washer;start
theprogram toto 30 ml 2x BF-BEB
Without Listex
Listex treated samples				without
shaking
andunder
to2x 30
ml 2x •BF-BEB
BF-BEB
42°C
Mix gently
microaerobic
conditions
without
shakingandunder
DNA extraction
and PCR analysis were performed as above.
Listeria andIC targets
weredeterminedand42°C
removethesupernatant
No pretreatment
Not activated FDRS B
Activated FDRS B
Activated FDRS D
2x BF-BEB• Mix gently
microaerobic
2x BF-BEB conditions• Mix gently • Incubate for 24 hrs at 42°C microaerobic conditions
sensitivity was estimated accordingly.
* Removepossible
fat layers
using
a tip
without
shakingand byunder			Mean of Listeria spp. 		
Mean of Listeria spp.		
Mean of Listeria spp.		
Mean of Listeria spp.

• Incubate for 24 hrs at 42°C
without shaking and underconditionsµl
Figure 1. Modified iQ-Check Listeria spp.microaerobic
assay involving
the Free DNA Removal 200
protocol10 µl

100 µlEnriched
sample10-15 min

Activated
FDRS

95-100°C

suspension• Add 100 µl of the lysis reagent(reagentA) enrichment
in the deepwell plate
Spike level
before6.4 CFU/25g• AddLysis
200reagentµl of thecompletelysis
reagent(reagent+ suspension
reagent F) to100 µlmust
be constantly
stirring
in orderto keep A
it in
each
well byusing
micropipette
10-1tips• Transfer
100µl of aenrichedsamplewith large opening 200 µl filter
Bacteria lysis +• Add 100 µl of
the
lysis
reagent(reagentA)
inthe
deepwellplate• Add 100 µl of the lysis reagent (reagent A)
in the deepwell plate• Seal
thethedeepwell
pre-pierced
sealing
10-2• Sealdeepwellplate
plate usingwith thea pre-pierced
sealingfilm tape
FDRS inactivation

Free DNA removal

100 µlreagent
must
be constantly stirringin orderoftoenrichement
keep it in suspension
10-fold dilutions
Lysis reagent must be constantly
stirring in order to keep itLysis
in suspension
DNA
extracted
10-3
from
intactcells sample•
Transfer
100µlofenriched
1010-4• Transfer 100 µl of enriched sample
3 min ± 1 min
15–
20 min • Incubate the deepwell plate in a thermomixer at 99°C for 3 min ± 1 min
15min
1,300 rpm
15–20
min.
•
Incubate
at95-100°C
for
10-15
min
at
1,300
rpm
in
a
plate
agitator• Seal
the deepwell plate with the pre-pierced sealing film
10-5
1,300 rpmmin.
• Seal the15–30
deepwellplate with the pre-pierced sealing film
at 1,300
rpm
1,300
rpm
95incubator
95-100°C-6
99°C

37+/ -2ºC

100°C for 10-15 min

2 min
2,250 gCq values (10 replicates)

Positive result

Cq values (10 replicates)

Positive result

Cq values (10 replicates)

Positive result

Cq values (10 replicates)

0%
100%

N/A
28.27

100%
100%

32.57
28.72

0%
100%

N/A
29.61

50%
100%

37.35
37.59

100%
100%
80%
20%
10%
0%
0%

31.92
34.65
37.10
38.67
39.40
N/A
N/A

100%
100%
100%
100%
100%
100%
100%

32.32
32.33
32.37
32.38
32.21
32.23
32.37

100%
100%
70%
60%
40%
40%
20%

32.91
36.01
39.27
40.95
41.50
41.98
42.24

100%
100%
100%
100%
100%
100%
100%

37.67
37.62
37.28
37.80
38.39
38.03
37.57

probe
mix

• Prepareplate
the PCR mix• Cool the deepwell• Centrifuge
2,250
min microplate• Distributeat45µl/wellginfor
thePCR
It2wasobserved that treating cheese with Listex led to a DNA contamination of the samples. The contamination gave rise to a PCR signal in the 35-37 range detected by iQ-Check Listeria

amplification
mix

45 µl

45 µl

45 µl

• Prepare the PCR mix• Distribute 45 µl/wellin theprobe
PCR
microplate
probe
amplification
PCRamplification
PCR
mix

PCR
mix

Positive result

15–20 min• Incubate at 95-100°C for 10-15 min at 1,300 rpm in a plate agitator1,300 rpm• Incubate at 95-100°C for 10-15 min at95-100°C
1,300 rpm in incubatora plate agitatorConclusions
45 µl

All Real-Time PCR assays were run on the
CFX96 Touch™ Deep Well instrument.

amplification
mix

10
10-7

100°C
95–100ºC • Transfer• Cool theplatethedeepwelldeepwellplate in a dry heating block; incubate at 95-

Lysis buffer

incubator• Cool the deepwell plate

probe
mix

• Incubate for 24
hrs at 42°C
microaerobic
conditions		
without shaking and under• Lysis reagent must be constantly stirring in order to keep it in
Unspiked
0 CFU
microaerobic conditions

mix

mix mix

5 µl

mix

PCR
mix

spp. When pretreated with the Free DNA Removal Solution, the signal of the dead cells was readily suppressed whereas the signal of the living cells was not affected. Furthermore, theInternal Control signal was not impacted, confirming that the lysis step with the Bio-Rad proprietary buffer efficiently inactivated the enzymatic mix. We optimized an enzymatic mix yielding• Add 5 µl of controls
optimal results with a pre-incubation step at 37°C ± 2°C for 15 to 30 min prior to the extraction with the iQ-Check Listeria spp. PCR assay with neither additional extraction steps nor•PCR
Add 5mixµl of sample supernatants•5 µlPrepare •thePrepare
the
PCR
mix
(See
PCR mix
calculation guide)
supplementary
not vortex before collecting
the sample equipment. This reagent which is now available (Figure 2) can be adapted to different matrices/broth /pathogen combinations and is easily automatable.
• Distribute 45 Doµl/wellin the PCR microplate

Figure 2. iQ-Check
Free DNA Removal
Solution• Distributethe PCR(45 µl) in the PCR microplate
Check there
are nomix
bubbles• Seal the microplate

16-1119

mix

Addfoodscience.bio-rad.com
5 µl of controls
Scan this code to learn more or• visit
5 µl• Add 5 µl of sample supernatants
Do not vortex before collecting the sample
5 µl• Add 5 µl• of•controls
Start software
Transfer
5 µl of the supernatant by using the multichannel micropipette,•
Createtheplate setup
fromthe deepwellto the PCR microplate• Add 5 µl of sample
supernatants
Visit us at foodscience.bio-rad.com• Startthetheamplification
by µl)
clicking
on “Run”• Distributecontrols (5Do not vortex
beforecollecting
thesample• Checkare no bubbles
Check there
are there
no bubbles
•
Seal
the
microplate• Seal the microplate

Bio-Rad Laboratories, Inc. | Food Science Division | 2000 Alfred Nobel Drive | Hercules, CA 94547 USA