Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification
Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification
Sabine Fuchs-Telka 0 1
Susanne Fister 0 1
Patrick-Julian Mester 0 1
Martin Wagner 0 1
Peter Rossmanith 0 1
0 Institute for Milk Hygiene, Milk Technology, and Food Science, University of Veterinary Medicine , Veterinärplatz 1, 1210 Vienna , Austria
1 Christian Doppler Laboratory for Monitoring of Microbial Contaminants, University of Veterinary Medicine , Veterinärplatz 1, 1210 Vienna , Austria
DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced.
Ionic liquids; DNA extraction; Quantitative PCR; Salmonella Typhimurium; Escherichia coli; Solvent extraction; Two-phase system
In the life sciences, particularly in molecular biology, one of
the most important classes of molecules are nucleic acids,
especially DNA. Because of their prominent role in all known
living organisms, various methods have been developed to
analyze them to address biological and biochemical questions
in clinical diagnostics, food safety, genomics, microbiology,
and environmental science [1–7]. Today bioanalytical
techniques, including PCR, quantitative PCR (qPCR), cloning,
and sequencing methods are capable of analyzing very small
amounts of target molecules (in the case of qPCR even down
to one target molecule), which as a consequence must be of
high purity and quality [8, 9]. State-of-the-art DNA extraction
methods either are mostly column based or use functionalized
magnetic particles, which renders them laborious to perform
and difficult to automate .
Novel approaches utilizing ionic liquids (ILs) have also
been successfully applied to DNA extraction, further
improving the previously established systems, via a second
hydrophobic extraction phase, solid-phase microextraction, or
magnetic IL extraction [11–14].
What all these extraction methods have in common is that
they can be separated into two general steps: the first step is
lysis of the cell; the second step is separation and purification
whereby the desired nucleic acid is extracted from the aqueous
cell debris and sample residues. It is of the utmost importance
to differentiate between these two general steps in DNA
extraction as they are often incorrectly conflated; most commercial
and recently developed methods focus only on the extraction
step while ignoring cell lysis. In respect of the various
application fields, it has to be made clear that crude DNA extracts are
suitable for only very few applications, and in most cases
quantitative nucleic acid isolation is necessary [1, 10]. This is
especially true in diagnostics, where legal regulations define the
number of pathogens permitted to be present in the sample or
only a small number of target cells are present within a huge
background . This problem is very well reflected by the
actual time required for the two separate steps in commercial
DNA purification kits for bacteria, such as the NucleoSpin®
tissue kit, which was used in this study. According to the
manufacture’s specifications, the cell lysis step with the
NucleoSpin® tissue kit for bacteria takes between 2.5 and
20 h, whereas the actual silica-column-based DNA extraction
and purification takes only approximately 5 min per sample. In
this context, cell lysis is the most crucial step, and the time
required for its execution is crucial. Because of the
characteristics of ILs, such as their ability to dissolve biomass, their high
thermal stability, and their ability to preserve DNA integrity in
the presence of nucleases, they have recently been recognized
to improve DNA extraction steps, cell lysis, and purification.
To the best of our knowledge, hydrophilic ILs have been most
successful for cell lysis [15, 16], whereas hydrophobic ILs have
proven useful for extraction of DNA from crude cell extracts
[11, 17]. The downside of hydrophilic ILs is their possible
interference or even complete inhibition of subsequent
molecular-biological methods, such as qPCR , and
therefore must be removed before analysis, and quantitative
backextraction of DNA from hydrophobic ILs is also challenging.
We report here a new method, based on hydrophobic ILs,
for fast and quantitative bacterial cell lysis with subsequent
DNA extraction followed by downstream
molecularbiological techniques such as qPCR. A set of five
hydrophobic ILs were carefully evaluated for their potential as cell lysis/
DNA extraction liquids, and the whole method was further
tested and improved for the final candidate. The final method
permits quantitative DNA extraction from Gram-negative
bacteria with performance equivalent to that of a commercial
kit in only a fraction of the time.
Materials and methods
ILs, test kits, and chemicals
I L s N - b u t y l d i e t h a n o l a m m o n i u m b i s ( t r i f l u o r o
([BMPyr+][Ntf2-]), and 1-hexyl-3-methylimidazolium
and precoated cellulose thin-layer chromatography (TLC)
plates (20 × 20) were provided by Merck (Darmstadt,
Germany). The NucleoSpin® tissue kit was provided by
Macherey-Nagel (Düren, Germany).
Diethylpyrocarbonatetreated water, primer, and probes were provided by Invitrogen
(Lofer, Austria). MgCl2 (purity 98% or greater, CAS
7786-303), KCl (purity 99% or greater, CAS 7447-40-7), NaCl (purity
98% or greater, CAS 7647-14-5), amino acids as their L
enantiomers (alanine, cysteine, glutamine, histidine, leucine,
phenylalanine, tyrosine, and tryptophan, purity 99% or greater),
monosaccharides as their D enantiomers (glucose, fructose, mannose,
and galactose), and Taq polymerase were provided by Fisher
Scientific (Vienna, Austria).
Bacterial strains and culture conditions
Salmonella Typhimurium (NCTC 12023) and Escherichia
coli TOP10F were used as model organisms for
Gramnegative bacteria. Listeria monocytogenes EGDe (1/2a,
internal number 2964) was used as a model organism for
Grampositive bacteria. All strains were maintained at -80 °C with
use of MicroBank technology (Pro-Lab Diagnostics,
Richmond Hill, Canada). L. monocytogenes, Salmonella
Typhimurium, and E. coli TOP10F are part of the collection
of bacterial strains at the Institute of Milk Hygiene,
Department of Veterinary Public Health and Food Science,
University of Veterinary Medicine (Vienna, Austria). All
bacterial strains were grown overnight in tryptone soy broth with
0.6% (w/v) yeast extract (Oxoid, Basingstoke, UK) at the
optimal growth temperature of 37 °C.
Quantitative PCR setup and DNA standard preparation
An Mx3000 qPCR thermocycler (Stratagene, La Jolla, CA,
USA) was used for the qPCR experiments; the 25-μl reaction
mixture contained 5 μl DNA template. The qPCR results were
expressed as bacterial cell equivalents, and all qPCR assays were
performed in duplicate. Quantitative PCRs (qPCRs) for
quantification of L. monocytogenes , Salmonella Typhimurium
, and E. coli  were performed as previously reported,
and detailed information about the respective qPCR assays is
summarized in the electronic supplementary material.
One milliliter of an overnight culture of each bacterial
species was used for DNA isolation with the NucleoSpin® tissue
kit, according to the manufacturer ’s specifications.
Determination of DNA concentration was performed by
fluorimetric measurement with an Hoefer DyNA Quant 200
apparatus (Pharmacia Biotech, San Francisco, CA, USA).
Dilution series (two to five dilutions) in 1× PCR buffer were
made. These dilutions were quantified by qPCR, and the
series were treated as a standard curve. The efficiency and the R2
correlation statistic were calculated.
Testing of inhibition of qPCR by ILs and evaluation
of DNA distribution between aqueous and IL phases
We added 250 μL of double-distilled water (ddH2O) to 250 μl
of the respective ILs. After agitation (30 s) and centrifugation
(5 min, 2500 g) to obtain phase separation, the upper aqueous
phase was transferred into a new tube. To measure the
influence of solubilized or dispersed hydrophobic ILs in the
aqueous phase, 5 μl of the aqueous phases was added to the master
mix of the Salmonella Typhimurium qPCR already including
1 × 105 Salmonella Typhimurium DNA copies.
For evaluation of DNA distribution, 250 μL of a 5 × 10-3
ng/μl solution of Salmonella Typhimurium DNA solution was
added to 250 μl of the respective ILs. After agitation (30 s)
and centrifugation (5 min, 2500 g) to obtain phase separation,
the upper aqueous phase was transferred into a new tube. The
IL phase was again extracted with 250 μl ddH2O. The DNA
concentration in both aqueous phases was measured by qPCR.
Development of an apparatus for cell disruption
and experimental setup
As small-volume containers (100 μl or less) for ILs, 0.3-ml
glass microcartridges for screw thread bottles (40 mm ×
6 mm; Fisherbrand, Fisher Scientific Austria, Vienna,
Austria), equipment normally used for high-performance
liquid chromatography measurements, were used. For heating,
an aluminum block with drilled holes, fitting the dimensions
of the microcartridges, was custom-manufactured and placed
on a heating plate (IKAMAG® RCT; IKA-Labortechnik,
Staufen im Breisgau, Germany) (see Fig. S1). The
temperature was measured with a metal thermometer (Ama-digit ad 14
th, Amarell, Kreuzwertheim, Germany) directly in one
reference vial containing IL placed in the aluminum block.
For the cell disruption experiments, overnight cultures of
Salmonella Typhimurium or E. coli were harvested by
centrifugation (5 min, 6000 g), washed three times with ddH2O, and
resuspended in 1/20 of the initial volume in ddH2O. Ten
microliters of the resuspended culture was placed into 100 μl of
[BMPyr+][Ntf2-], covered with porous aluminum foil, and
incubated for various times (1, 2, 5, and 30 min) and at different
temperatures (80, 120, 150, and 180 °C). Following
incubation, 250 μl ddH2O was added, and the two phases were
mixed with a pipette and transferred into a 2-ml tube
(Eppendorf, Hamburg, Germany). An additional 250-μl
ddH2O aliquot was used as the vehicle to transfer the remnant
into the tube. The tube contents were briefly vortexed, and the
upper aqueous phase was used directly for qPCR
measurements following rapid, spontaneous IL separation despite not
introducing an additional centrifugation step.
Ten microliters of the same bacterial suspension used in the
cell disruption experiments was used for DNA isolation with
the NucleoSpin® tissue kit in accordance with the
manufacturer’s specifications. The final step of the protocol was
modified. Instead of one wash with 100 μl prewarmed (70 °C)
elution buffer BE, two washes with 50 μl prewarmed
ddH2O were used for DNA elution from the column .
Finally, 400 μl of ddH2O was added to achieve the same
volume as in the cell disruption experiments. The samples
were used directly for qPCR.
Extraction of salts, sugars, and lipids
Aqueous solutions of the chloride salts of magnesium
(30 ppm), potassium (300 ppm), and sodium (40 ppm) were
prepared and vortexed in a ratio of 1:1 with [BMPyr+][Ntf2-].
These solutions, including controls, were diluted 100-fold and
analyzed with a PerkinElmer 3030B atomic absorption
spectrometer (PerkinElmer, Wellesley, MA, USA), according to
the manufacturer’s instructions. Monosaccharide (D-glucose,
D-fructose, D-mannose, D-galactose) solutions were prepared
according to the protocol of Matissek et al. . Solutions
were either applied directly onto the TLC plate, vortexed in
a ratio of 1:1 with [BMPyr+][Ntf2-] before application, or
applied after an additional centrifugation step (5 min, 6000 g).
TLC plates were developed according to the protocol for the
identification of sugars by Matissek et al. . Amino acid
solutions (L-alanine, L-cysteine, L-glutamine, L-histidine,
Lleucine, L-phenylalanine, L-tyrosine, and L-tryptophan) were
prepared according to the method of Matissek et al. .
Either amino acid solutions were applied directly or a mixture
of all eight amino acids was vortexed in a ratio of 1:1 with
[BMPyr+][Ntf2-] with and without additional centrifugation
(5 min, 6000 g) or preincubated (2 min, 140 °C) in a ratio of
1:1 with [BMPyr+][Ntf2-] with and without additional
centrifugation for (5 min, 6,000 g). The TLC plates were developed
according to the protocol for the identification of amino acids
by Matissek et al. .
Transmission electron microscopy
The protocol of Glauert et al.  was followed to prepare
negative-stained ultrathin section samples. In brief, prefixation
was achieved with 5% glutaraldehyde, and fixation was
achieved with 1% osmium tetroxide. Samples were mixed with
agar, dehydrated, and embedded. After polymerization, ultrathin
sections with a thickness of 70–90 nm were cut, and staining of
the samples for transmission electron microscopy was
performed with phosphotungstic acid . Negative-stained
ultrathin section samples of Salmonella Typhimurium and E. coli
were analyzed with a Zeiss (Vienna, Austria) EM900
transmission electron microscope after the respective cell disruption
procedures had been conducted.
Results and discussion
The development of an efficient cell lysis method for
quantitative pathogen detection by the five hydrophobic ILs required
evaluation of their DNA uptake capacity and their possible
capacity for inhibition of downstream methods. In contrast
to previously studied IL-based DNA extraction methods, the
DNA intake capacity of the ILs should preferably be very
small to minimize the need for multiple elution steps
following cell lysis, and thereby reduce the need for complex
handling and shorten the time required. Hydrophobic ILs often
consist of cations possessing long alkyl side chains and/or of
fluorinated anions, both which have been previously reported
to interfere or inhibit enzymatic reactions such as PCR [17,
18]. Although several possible intervention strategies have
recently been reported to protect PCR-based methods from
inhibition by hydrophobic ILs [15, 17], limited solubility of
ILs in the aqueous elution phase would be preferable.
Quantitative PCR inhibition by hydrophobic ILs
in the aqueous eluate
To investigate the applicability of the five hydrophobic ILs
used in this study, IL-mitigated inhibition was tested directly
with use of qPCR. To simulate extraction, the respective ILs
were agitated for 30 s together with an equal volume of
ddH2O, and after separation via centrifugation (5 min at
2500 g), the aqueous phase was tested for possible
interference or inhibition of a Salmonella Typhimurium qPCR. In the
case of [DEBA+][Ntf2-] and [C6mim+][FAP-], complete
inhibition of the qPCR was observed when the aqueous phase,
separated after extraction, was applied directly (Fig. 1, plots A
and B). To prevent inhibition by these two ILs, it was
necessary to make a tenfold dilution of the aqueous phase (Fig. 1,
plot C). The respective aqueous phases for [N6,6,6,14+][FAP-]
and [N6,6,6,14+][Ntf2-] resulted in a reduced qPCR
efficiency of approximately 80%, indicating a slight
inhibition of the enzymatic reaction due to the presence of IL.
The only hydrophobic IL that did not cause any qPCR
inhibition was [BMPyr+][Ntf2-], and therefore this IL
could be used without additional purification or
Fig. 1 Quantitative PCR (qPCR) inhibition of aqueous phases caused by
leaching of hydrophobic ionic liquids compared wit a control (A) and
mean values with the respective standard deviation of three independent
experiments (B). Dilutions of the aqueous phases extracted from
( [ N 6 , 6 , 6 , 1 4 + ] [ FA P - ] ) a n d t r i h e x y l t e t r a d e c y l p h o s p h o n i u m
bis(trifluoromethylsulfonyl)imide ([N6,6,6,14+][Ntf2-]) in double-distilled
water (ddH2O) and their respective mean inhibition values with the
respective standard deviation of the qPCR (C). [Bmpyr+][Ntf2-]
[ C 6 m i m + ] [ F A P - ] 1 - h e x y l - 3 - m e t h y l i m i d a z o l i u m
tris(pentafluoroethyl)trifluorophosphate, [DEBA+][Ntf2-] N-butyl
diethanol ammonium bis(trifluoromethylsulfonyl)imide
DNA uptake by hydrophobic ILs
As for the inhibition studies, genomic DNA of the
Gramnegative bacterium Salmonella Typhimurium was selected as a
model to measure the IL DNA uptake capacity. DNA in aqueous
solution (5 × 10-3 ng/μl) was agitated for 30 s together with an
equal volume of a particular IL. The respective DNA
concentration in the aqueous phase directly after phase separation and after
a second extraction step was determined by qPCR, and the
results are shown in Fig. 2. Because of the inhibitory effects of IL
remnants as determined in the previous experiments, respective
aqueous solutions of [N6,6,6,14+][FAP-], [N6,6,6,14+][Ntf2-],
[C6mim+][FAP-], and [DEBA+][Ntf2-] were diluted tenfold in
ddH2O. For [N6,6,6,14+][FAP-] and [DEBA+][Ntf2-], an almost
stoichiometric distribution of the genomic DNA between the IL
phase and the aqueous phase was determined, which is in good
accordance with the findings of previous studies using
hydrophobic ILs to extract DNA from aqueous solution [11, 13, 17].
However, although desirable in such applications, DNA uptake
is disadvantageous during lysis of bacterial cells as quantitative
release of DNA from the IL phase is not possible, even after an
additional extraction step. For [N6,6,6,14+][Ntf2-] and
[C6mim+][FAP-], almost no DNA uptake into the IL phase was
measured. However, as discussed, following extraction, for both
ILs the aqueous phase had to be diluted to prevent qPCR
inhibition secondary to IL in the aqueous eluate. No DNA uptake into
the IL phase was observed for [BMPyr+][Ntf2-], and in contrast
Fig. 2 DNA quantity in the aqueous phase after extraction with ionic
liquids compared with the initial input. Mean values with the respective
standard deviation of three independent experiments are shown. The
number 2 indicates the second extraction cycle. Samples that had to be
diluted tenfold in ddH2O to avoid qPCR inhibition are indicated by an
asterisk. rel. relative
to the other ILs tested, the aqueous phase following extraction
could be used directly without additional dilution.
Uptake of cellular components by [BMPyr+][Ntf2-]
Although the main focus of this study was to investigate
hydrophobic ILs for bacterial cell lysis and DNA extraction,
secondary goals included the possibility of DNA purification
from cellular debris and the extent of downstream inhibition.
Hydrophobic ILs have been previously reported to take up
selectively and thus purify DNA from crude cellular extracts
[13, 19]. As previously indicated, for the proposed application
of cell lysis and DNA extraction, selective DNA uptake would
be disadvantageous. Nevertheless, although [BMPyr+][Ntf2-]
was specifically selected because of its negligible DNA
uptake, possible uptake of cellular components such as salts,
lipids, carbohydrates, or proteins (all of which can inhibit
downstream molecular-biological methods) was also
analogously investigated. The presence of salts (i.e., KCl, NaCl,
MgCl2) was analyzed by electrothermal atomic absorption
spectroscopy (ET-AAS). The concentrations to be measured
were chosen to be at least 100-fold above the limit of
quantification of the ET-AAS device (300 ± 3 ppb for Mg; 2986 ±
5 ppb for K; 383 ± 6 ppb for Na). The results demonstrated no
removal from the aqueous phase after phase extraction with
[BMPyr+][Ntf2-] (Mg, 294 ± 3 ppb; K, 2972 ± 13 ppb; Na, 381
± 6 ppb). For carbohydrates, lipids, and proteins, TLC
methods were used for semiquantitative evaluation.
Likewise, in the case of monosaccharides (glucose, fructose,
galactose, and mannose), amino acids (see Figs. S3, S4), and
lipids (results not shown), there was no uptake by
[BMPyr+][Ntf2-] of any of the substances tested.
For actual disruption of cells, several approaches are commonly
used, and can be classified as physical (e.g., boiling, grinding,
osmotic shock, dry–thaw), chemical (e.g., chaotropic or
chelating agents, detergent lysis, acid/base), or biological (e.g.,
enzymes such as lysozyme or proteinase K, phages) methods.
Each of these methods has certain advantages and
disadvantages , but for automation and to reduce the number of
handling steps, either boiling or chemical lysis or a
combination of both is most advantageous. Although boiling in water
has been reported to be efficient for the extraction of nucleic
acids from eukaryotic cell cultures, this technique does not lead
to quantitative release from more complex matrices or
especially bacterial cells. One of the main advantages of ILs in this
regard is, of course, their excellent thermal stability and
negligible vapor pressures, which permits the use of higher
temperatures than would be possible with aqueous solutions . Of
the five hydrophobic ILs investigated, only [BMPyr+][Ntf2-]
did not show considerable DNA uptake (which would result in
a quantitative loss) or qPCR inhibition by inevitable remnants
of this IL in the aqueous phase. Therefore it can be considered
the ideal candidate to investigate the possible application of ILs
as DNA extraction/lysis liquids.
After the establishment of a device and protocol feasible for
efficient use at elevated temperatures (detailed information is
available in the electronic supplementary material), the efficiency
of bacterial cell lysis with changing incubation times and
temperatures was investigated. To assess the general applicability of
the new method, three different bacterial species (Gram-negative
E. coli and Salmonella Typhimurium, and Gram-positive
L. monocytogenes) were used, and the DNA lysis efficiency by
qPCR was quantitatively compared with that of the
commercially available NucleoSpin® tissue kit. Control samples prepared
with the commercial kit were treated according to the
manufacturer’s recommendations to achieve approximately 90% cell
lysis efficiency, with a total protocol time of 3 h for E. coli and
Salmonella Typhimurium and 20 h for L. monocytogenes.
The results presented in Fig. 3 show that the newly
developed hydrophobic-IL-based method is able to achieve the
Fig. 3 Time–temperature diagrams of the cell lysis protocol with
[BMPyr+][Ntf2-]. For Escherichia coli (A) the highest value was
measured at 1 min and 150 °C ,and for Salmonella Typhimurium (B) it
was measured at 1 min and 120/150 °C. All data were calculated in
proportion to the highest measured value. All samples were prepared in
duplicate and a mean was calculated
same quantitative cell lysis values for both E. coli and
Salmonella Typhimurium in 1 min as the commercial kit
achieves in 3 h, whereas quantitative DNA extraction was
not possible from L. monocytogenes (data not shown). For
both E. coli and Salmonella Typhimurium, the optimal
temperature for efficient cell lysis was between 120 and 150 °C.
Although lower temperatures resulted in lower quantitative
cell lysis efficiency, especially for E .coli, the highest tested
temperature (180 °C) also resulted in lower values, most likely
due to DNA thermal degradation. The incubation time,
surprisingly, did not play a major role in terms of extraction
efficiency. Indeed, the only effect observed was a loss of
DNA extraction efficiency at elevated temperatures, again
probably due to thermal DNA degradation.
To evaluate the purity grade of the extracted nucleic acids with
regard to inhibitory effects on subsequent methods (e.g., qPCR,
cloning, restriction analysis), dilution series were compared with
conventionally extracted nucleic acids (NucleoSpin® tissue kit)
in qPCR. As the qPCR system is one of the most sensitive
molecular methods, it was assumed that other methods should
not be inhibited. As quality parameters, efficiency and R2 of the
standard curve were compared, and similar values were obtained
for both bacteria by the newly developed method (see Table 1).
In contrast to the promising results obtained by the new
method for E. coli and Salmonella Typhimurium, only
approximately 5% cell lysis efficiency in comparison with the
commercial kit was obtained for L. monocytogenes. The most
probable explanation for these significantly different results
reflects the differences between the cell envelopes of
Gramnegative and Gram-positive bacteria. Whereas the cell
envelope of Gram-negative bacteria comprises two cellular lipid
bilayer membranes and only a small cell wall comprising
juxtaposed peptidoglycan, the cell envelope of Gram-positive
bacteria is characterized by a single membrane and a much
thicker outer cell wall. It is generally recognized that the thick
cell wall of Gram-positive bacteria protects cellular integrity
against physical and chemical stresses more effectively than
the cell envelope of Gram-negative bacteria. This is also
Table 1 Mean cell lysis efficiency and R2 were calculated from six
independent measurements and a series of three to five dilutions was
Cell lysis efficiency R2
Salmonella Typhimurium Conventionala 96.53
IL extraction 98.63
Escherichia coli Conventionala 94.26
IL extraction 88.08
Fig. 4 Transmission electron microscopy micrographs after the different
extraction protocols with [BMPyr+][Ntf2-]. Micrographs of Salmonella
Typhimurium (a), E. coli (b), and Listeria monocytogenes (c) controls.
For the final extraction protocol, 1 min and 150 °C were selected for all
three species (d Salmonella Typhimurium, e E. coli, and f
L. monocytogenes). Bars 500 nm
acknowledged for the NucleoSpin® tissue kit, the instructions
for which recommend an additional overnight enzymatic
digestion step in its support protocol for Gram-positive bacteria.
To verify the influence on the cell membrane as well as the cell
wall, transmission electron microscopy micrographs of the
three bacterial species before and after the incubation step
with [BMPyr+][Ntf2-] were obtained (Fig. 4). It can be
observed that Salmonella Typhimurium and E. coli are
completely disrupted by [BMPyr+][Ntf2-], whereas the
cellular integrity of L. monocytogenes was less affected.
In summary, a novel method based on hydrophobic ILs was
systematically developed for bacterial cell lysis and quantitative
DNA extraction for downstream molecular-biological analysis.
The use of hydrophobic ILs permitted the use of elevated
temperatures between 120 and 150 °C that are required for complete
disintegration of Gram-negative bacteria cells. However, under
these conditions, a Gram-positive species was still protected by a
thick cell wall. Although no purification of genomic DNA from
cellular constituents, such as carbohydrates, lipids, or salts,
occurred, DNA extracted with the new protocol was of such high
quality that subsequent molecular-biological methods (e.g.,
qPCR) could be readily used without further purification steps.
This study demonstrates that hydrophobic-IL-based DNA
extraction resulted in quantitative DNA extraction efficiencies for
Gram-negative bacteria similar to those obtained with a
commercial kit, whereas the number of handling steps, and especially the
time required, was dramatically reduced. In combination with
recently developed DNA concentration and purification
techniques, such as magnetic IL extraction or aqueous biphasic
systems, the new method has the potential to shorten significantly
the time required for clinical and food diagnostics as well as
Acknowledgements Open access funding provided by University of
Veterinary Medicine Vienna. Financial support by the Austrian Federal
Ministry of Science, Research and Economy and the National
Foundation of Research, Technology and Development is gratefully
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Sabine Fuchs-Telka is a research
associate in the Christian Doppler
Laboratory for Monitoring of
Microbial Contaminants at the
U n i v e r s i t y o f Ve t e r i n a r y
Medicine in Vienna, Austria. She
is working on the development of
new cell lysis and DNA extraction
methods for isolation of DNA
from prokaryotic cells based on
Susanne Fister is a postdoctoral
research fellow in the Christian
D o p p l e r L a b o r a t o r y f o r
M o n i t o r i n g o f M i c r o b i a l
Contaminants at the University
o f Ve t e r i n a r y M e d i c i n e i n
Vienna, Austria. She is working
with phages and viruses relevant
in the food industry (phages P100
and MS2, feline and murine
noroviruses), and studies the use
of ionic liquids for the
development of new methods and
applications in (food) microbiology.
Patrick-Julian Mester is a
postdoctoral research fellow in the
Christian Doppler Laboratory for
M o n i t o r i n g o f M i c r o b i a l
Contaminants at the University
o f Ve t e r i n a r y M e d i c i n e i n
Vienna, Austria. His main
research interests are studying the
interaction and possible
application of ionic liquids in the field
of biotechnology and
microbiology, as well as the development of
novel detection methods for
Martin Wagner is Head of the
Institute for Milk Hygiene, Milk
Technology, and Food Science in
the Department for Farm Animals
and Veterinary Public Health at
the University of Veterinary
Medicine in Vienna, Austria. His
focus is the differentiation,
detection, and phylogenetic analyses of
microbial pathogens along the
Peter Rossmanith is Head of the
Christian Doppler Laboratory for
M o n i t o r i n g o f M i c r o b i a l
Contaminants in the Department
for Farm Animals and Veterinary
Public Health at the University of
Veterinary Medicine in Vienna,
Austria. His focus is on the
development of methods for detection
and analysis of microbes and the
integration of ionic liquids in
1. Jaykus LA . Challenges to developing real-time methods to detect pathogens in foods . Asm News . 2003 ; 69 : 341 - 7 .
2. Bustin SA , Mueller R. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis . Clin Sci . 2005 ; 109 : 365 - 79 .
3. Cho I , Blaser MJ . The human microbiome: at the interface of health and disease . Nat Rev Genet . 2012 ; 13 : 260 - 70 .
4. Ivnitski D , Abdel-Hamid I , Atanasov P , Wilkins E. Biosensors for detection of pathogenic bacteria . Biosens Bioelectron . 1999 ; 14 : 599 - 624 .
5. Petrosino JF , Highlander S , Luna RA , Gibbs RA , Versalovic J. Metagenomic pyrosequencing and microbial identification . Clin Chem . 2009 ; 55 : 856 - 66 .
6. Velusamy V , Arshak K , Korostynska O , Oliwa K , Adley C. An overview of foodborne pathogen detection: in the perspective of biosensors . Biotechnol Adv . 2010 ; 28 : 232 - 54 .
7. Voelkerding KV , Dames SA , Durtschi JD . Next-generation sequencing: from basic research to diagnostics . Clin Chem . 2009 ; 55 : 641 - 58 .
8. Rossmanith P , Wagner M. The challenge to quantify Listeria monocytogenes - a model leading to new aspects in molecular biological food pathogen detection . J Appl Microbiol . 2010 ; 110 : 605 - 17 .
9. Witte AK , Fister S , Mester P , Schoder D , Rossmanith P. Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR . Anal Bioanal Chem . 2016 ; 408 ( 27 ): 7583 - 93 .
10. Kauffmann IM , Schmitt J , Schmid RD . DNA isolation from soil samples for cloning in different hosts . Appl Microbiol Biotechnol . 2004 ; 64 : 665 - 70 .
11. Wang JH , Cheng DH , Chen XW , Du Z , Fang ZL . Direct extraction of double-stranded DNA into ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate and its quantification . Anal Chim Acta . 2007 ; 79 : 620 - 5 .
12. Clark KD , Nacham O , Yu H , Li T , Yamsek MM , Ronning DR , et al. Extraction of DNA by magnetic ionic liquids: tunable solvents for rapid and selective DNA analysis . Anal Chem . 2015 ; 87 : 1552 - 9 .
13. Li T , Joshi MD , Ronning DR , Anderson JL . Ionic liquids as solvents for in situ dispersive liquid-liquid microextraction of DNA . J Chromatogr A. 2013 ; 1272 : 8 - 14 .
14. Nacham O , Clark KD , Anderson JL . Extraction and purification of DNA from complex biological sample matrices using solid-phase microextraction coupled with real-time PCR . Anal Chem . 2016 ; 88 : 7813 - 20 .
15. Fister S , Fuchs S , Mester P , Kilpeleinen I , Wagner M , Rossmanith P. The use of ionic liquids for cracking viruses for isolation of nucleic acids . Sep Purif Technol . 2015 ; 155 : 38 - 44 .
16. Ressmann AK , Garcia EG , Khlan D , Gaertner P , Mach RL , Krska R , et al. Fast and efficient extraction of DNA from meat and meat derived products using aqueous ionic liquid buffer systems . New J Chem. 2015 ; 39 : 4994 - 5002 .
17. Clark KD , Yamsek MM , Nacham O , Anderson JL . Magnetic ionic liquids as PCR-compatible solvents for DNA extraction from biological samples . Chem Commun . 2015 ; 51 : 16771 - 3 .
18. Naushad M , Alothman ZA , Khan AB , Ali M. Effect of ionic liquid on activity, stability, and structure of enzymes: a review . Int J Biol Macromol . 2012 ; 51 : 555 - 60 .
19. Rossmanith P , Krassnig M , Wagner M , Hein I. Detection of Listeria monocytogenes in food using a combined enrichment/real-time PCR method targeting the prfA gene . Res Microbiol . 2006 ; 157 : 763 - 71 .
20. Mann E , Hein I , Mester P , Stessl B , Rossmanith P , Wagner M , et al. A robust and poisson validated quantitative nuclease 5′ TaqMan® real-time PCR assay targeting fimA for the rapid detection of Salmonella spp . in food. Food Anal Methods . 2013 ; 6 : 991 - 5 .
21. Kaclikova E , Pangallo D , Oravcova K , Drahovska H , Kuchta T. Quantification of Escherichia coli by kinetic 5-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene . Lett Appl Microbiol . 2005 ; 41 : 132 - 5 .
22. Mayrl E , Roeder B , Mester P , Wagner P , Rossmanith P. Broad range evaluation of the matrix solubilization (matrix lysis) strategy for direct enumeration of food-borne pathogens by nucleic acids technologies . J Food Prot . 2009 ; 72 : 1225 - 33 .
23. Matissek RS , Fischer M. Lebensmittelanalytik . Berlin: Springer; 2010 .
24. Glauert AM , Lewis PR . Biological specimen preparation for transmission electron microscopy . Princeton: Princeton University Press ; 1999 .
25. Radstrom P , Knutsson R , Wolffs P , Lovenklev M , Lofstrom C. PrePCR processing: strategies to generate PCR-compatible samples . Appl Microbiol . 2004 ; 26 : 133 - 46 .
26. Huddleston JG , Visser AE , Reichert WM , Willauer HD , Broker GA , Rogers RD . Characterization and comparison of hydrophilic and hydrophobic room temperature ionic liquids incorporating the imidazolium cation . Green Chem . 2001 ; 3 : 156 - 64 .