Food Control 109 (2020) 106952

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Food Control
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Nucleic acid lateral flow immunoassay (NALFIA) with integrated DNA probe T
degradation for the rapid detection of Cronobacter sakazakii and Cronobacter
malonaticus in powdered infant formula
Ömer Akinedena,∗, Tobias Wittwerb, Katrin Geisterb, Madeleine Plötzc,1, Ewald Uslebera
a
Chair of Dairy Science, Institute of Veterinary Food Science, Justus-Liebig University Giessen, Ludwigstraße 21, D-35390, Giessen, Germany
b
R-Biopharm AG, An der Neuen Bergstraße 17, D-64297, Darmstadt, Germany
c
Junior Professorship of Veterinary Food Diagnostics, Institute of Veterinary Food Science, Justus-Liebig University Giessen, Ludwigstraße 21, D-35390, Giessen, Germany

ARTICLE INFO ABSTRACT

Keywords: A novel nucleic acid lateral flow immunoassay (NALFIA) system for Cronobacter (C.) sakazakii and C. malonaticus is
Cronobacter sakazakii based on amplification of the rpoB gene in the presence of a heterobifunctional, Cronobacter-specific DNA probe.
Cronobacter malonaticus Partial degradation of this probe during amplification is then detected by lateral flow immunoassay. The NALFIA
Enterobacter sakazakii detected all of the 22 C. sakazakii and 8 C. malonaticus isolates under study, but showed no reaction with other
Powdered infant formula
Cronobacter species or non-Cronobacter species. The minimal visual and instrumental detection limits were 104 cfu/
Nucleic acid lateral flow immunoassay
Rapid detection
ml for both species in buffered peptone water and in reconstituted infant formula. C. sakazakii or C. malonaticus at
levels of 100-101 cfu/g in powdered infant formula were reliably detected by NALFI after reconstitution and 24 h
incubation at 37 °C. A major advantage of this approach is that the use of an integrated DNA probe provides
additional test specificity. Since C. sakazakii and C. malonaticus are by far the most important Cronobacter species
occurring in PIF, the novel NALFIA is a promising tool to enhance rapid control of infant formula.

1. Introduction
Cronobacter spp. (formerly Enterobacter (E.) sakazakii), members of
the Enterobacteriaceae family (Iversen et al., 2008), are opportunistic
pathogens which have been associated worldwide with rare but life-
threatening disease (meningitis, septicaemia, necrotizing enterocolitis)
in newborn and premature infants. Currently the genus Cronobacter is
composed of seven recognized species, but only strains of three species,
C. sakazakii, C. malonaticus, and C. turicensis have been associated with
clinical infections in infants (Forsythe, 2018). C. sakazakii and C. mal-
onaticus are the most common Cronobacter species associated with
human disease (Holý & Forsythe, 2014) and are responsible for the
majority of Cronobacter infections in infants (Joseph et al., 2012). C.
sakazakii and C. malonaticus are the predominant Cronobacter species in
powdered infant formulae (PIF) worldwide (Akineden, Heinrich, Gross,
& Usleber, 2017; Yang et al., 2016), including cases in which PIF have
been identified as a source of infection for infants (Block et al., 2002;
Holý & Forsythe, 2014; Mullane et al., 2007; Van Acker et al., 2001).
Although the minimal infectious dose is not known, Cronobacter has
been found at levels less than 1 cfu per 100 g in PIF associated with
infections, therefore European Union regulation on microbiological
criteria for foodstuffs (European Commission, 2005) requires absence of
Cronobacter spp. in 30 × 10 g of PIF intended for special medical pur-
poses for infants under 6 months of age. The reference method for this
criterion is ISO/TS 22964:2006, which has recently been updated by
ISO 22964:2017 (De Benito et al., 2019). This method is both laborious
and time-consuming, and does not allow differentiation between spe-
cies. Although the reference method is mandatory for all legal purposes,
PIF production facilities require suitable additional means for a more
rapid presence/absence testing of relevant food safety parameters.
In response to the needs of food industry, several complementary
techniques have been established, either to obtain results more rapidly,
or to enable species differentiation. These include molecular detection
methods such as real time PCR (Yan & Fanning, 2015; Zhou et al., 2016;
Zimmermann, Schmidt, Loessner, & Weiss, 2014), fluorescence in situ
hybridization (Almeida et al., 2009), DNA microarray (Wang et al.,
2009), and immunoassays (Blažková, Javůrková, Fukal, & Rauch, 2011;
Park et al., 2012; Scharinger, Dietrich, Wittwer, Märtlbauer, & Schauer,

Corresponding author.
∗

E-mail address: Oemer.Akineden@vetmed.uni-giessen.de (Ö. Akineden).

1
Present Address: Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173, Hannover,
Germany

https://doi.org/10.1016/j.foodcont.2019.106952
Received 9 July 2019; Received in revised form 10 October 2019; Accepted 12 October 2019
Available online 15 October 2019
0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
Ö. Akineden, et al.
2017; Scharinger, Dietrich, Kleinsteuber, Märtlbauer, & Schauer, 2016;
Song, Shukla, Oh, Kim, & Kim, 2015; Xu et al., 2014).
A relatively new approach combines PCR amplification with im-
munochemical detection, most appropriately as lateral flow dipstick
tests for rapid detection. These methods combine the power of ampli-
fication of the target gene sequence with the sensitivity and ease of use
offered by dipstick immunoassay techniques. A promising variant of
such methods is NALFIA (Singh, Sharma, & Nara, 2015), which has
been successfully applied to the detection of e.g. Cronobacter spp.
(Blažková et al., 2011), Listeria monocytogenes (Blažková, Koets, Rauch,
& van Amerongen, 2009), methicillin-resistant Staphylococcus aureus
(Seidel et al., 2017; Zhang et al., 2017), Mycobacterium tuberculosis-
Complex (Haridas, Thiruvengadam, & Bishor, 2014) and Toxoplasma
gondii infections (Wu et al., 2017). These methods typically use 5′-end
labelled primers pairs with different labels for forward and reverse
primers. For example, Blažková et al. (2011) used biotin and digox-
igenin to label the 5′-end of the forward and reverse primers, respec-
tively, the doubly labelled amplicon was then visualized on a lateral
flow system in an anti-digoxigenin carbon-neutravidin sandwich com-
plex. This approach was found convenient and sensitive for detection of
Cronobacter at genus level. Here we report evaluation of a novel NALFIA
technique which employs a heterobifunctional DNA probe which binds
to a specific template DNA locus of C. sakazakii and C. malonaticus
framed by the primer pair used for amplification. The degradation
products of this probe, produced during the amplification in a single-
step reaction, are then measured by lateral flow immunoassay. Eva-
luation parameters included sensitivity, specificity and test robustness
when applied to detect C. sakazakii and C. malonaticus in PIF.
2. Materials and methods
2.1. Bacterial strains
The bacterial strains used in this study are listed in Tables 1 and 2
and were all from the institute's strain collection. The selection of
Cronobacter strains was based on origin and specific characteristics such
as species/subspecies, biotype, and serotype (Akineden et al., 2017).
For exclusivity testing, 21 non-Cronobacter strains belonging to dif-
ferent bacterial families and genera were used.
2.2. Test principle and components of NALFIA with integrated DNA probe
degradation
The test principle of the NALFIA with integrated DNA probe de-
gradation (Fig. 1) is based on amplification of the rpoB gene of C. sa-
kazakii or C. malonaticus in the presence of a heterobifunctional DNA
probe labelled with two different haptens, digoxigenin on the 5′ end and
biotin on the 3′ end. This probe binds to a specific template DNA locus of
C. sakazakii and C. malonaticus, framed by the primer pair used for
conventional PCR amplification. The degradation products of this probe,
produced during the amplification step, are then measured by lateral
flow immunoassay via antibodies against the hapten label. In positive
samples, the probe binds to DNA in a region framed by the forward and
reverse primers, but is then partially degraded by exonuclease during
amplification. The concentration of reagents was selected in such a way
that only a part of the probe is degraded during the PCR process,
therefore the intact portion of the probe could be used as an internal
control. The degradation product is visualized on a lateral flow dipstick
format (Fig. 1) which is typically composed of three parts: a sample pad
for loading of the sample, a reaction nitrocellulose membrane for for-
mation of the detectable colored lines and finally, an absorbent pad
enhancing the capillary driving force and adsorbing non-reacting sub-
stances. The membrane has two reaction zones, one for specific detection
of degraded probe, one as the control line to identify a valid test. On the
NALFIA test strips, intact probe reacts with gold-labelled anti-digox-
igenin monoclonal antibodies (DIG-mAb) and is visualized on a
2
Food Control 109 (2020) 106952
streptavidin-coated control field. In positive tests, the digoxigenin-la-
belled fragment of degraded probes, after binding to DIG-mAb, are fi-
nally visualized on a test field coated with anti-mAb. Test evaluation can
be performed either by visual estimation of color development or by
instrumental reading of the reflectance of reaction fields.
2.3. Primer and labelled DNA probe design
Primer pairs, DNA probe and strips were prepared and customized as
needed for this study in the laboratories of R-Biopharm AG, Germany.
The specific PCR primers and the DNA probe, targeting a highly con-
served region of rpoB gene of C. sakazakii and C. malonaticus, were de-
signed using PrimerExplorer V4 software (http://primerexplorer.jp/
elamp4.0.0/index.html) according to the sequence data as published in
the National Center for Biotechnology (NCBI) GenBank, USA. All primers
and probe were blasted against the NCBI nucleotide database to make
sure there was no homology with sequences from other organisms. The
final set included forward primer designated CsakFmm, reverse primer
designated rpoB-RVmm, producing an expected amplicon size of 210 bp.
The DNA probe, designated as rpoB-SNDn, was labelled with digoxigenin
at the 5′-end, and with biotin at the 3′end (Table 3), to provide a het-
erobifuntional label.
2.4. PCR amplification and control analysis of PCR products by agarose gel
electrophoresis
The PCR assay was carried out in a total of 25 μl reaction mixture
containing: 1 μl of each primer (1 μmol; CsakFmm and rpoB-RVmm),
1 μl of labelled DNA probe (0.1 μmol; rpoB-SNDn), 12.5 μl of a com-
mercial PCR master mix buffer/reagent solution (SensiFAST Probe No-
ROX 2x; Bioline, Berlin, Germany), and 9.5 μl sterile distilled water.
Finally, 2.5 μl of DNA template was added to each tube. The cycling
amplification was conducted on a Bio-Rad T100 Thermocycler (Bio-
Rad, Munich, Germany) according to the following steps: initial in-
cubation at 94 °C for 5 min, followed by 35 cycles of 95 °C for 20 s, 65 °C
for 45 s. After classical PCR amplification of the target genes, the PCR
amplification products were subjected to agarose gel electrophoresis, as
control analyses, parallel to the NALFIA procedure. The PCR products
(10 μl) were separated on 1.5% agarose gels, followed by ethidium
bromide staining and photography under UV light using the GelDoc
system (BioRad). DNA molecular size standards (100 bp gene ruler,
Fermentas) were included in each agarose gel. For the positive control
reaction, plasmid conjugated rpoB sequence was used. Lysis buffer was
used as for negative control reaction.
2.5. Lateral flow immunoassay
After amplification, 10 μl of the PCR reaction mixture and 90 μl of
NALFIA running buffer (45 mmol Bis-Tris, pH 7.5, 100 mmol NaCl;
0.05% Triton X-100; 0.01% sodium azide) were pipetted in a sterile
reaction tube and mixed gently using a pipette. Then the test strip was
immersed, sample pad first, into the test solution, allowing the liquid to
migrate through the dipstick membrane for about 12 min. After this
time, the test was completed and a red colored line was discernible for
the control field. The color intensities of the control and test lines were
evaluated visually by at least two persons. The presence of two, clearly
discernible red lines indicated a positive results for C. sakazakii and/or
C. malonaticus, while presence of one line (control line) indicated a
negative result. In case of invalid test results, neither test nor control
lines or only the test line were visible.. After visual evaluation, the
NALFIA strips were scanned and the reflectance of the test line quan-
titatively evaluated using a lateral flow reader (ESEQuant Lateral Flow
System, Qiagen, Germany) in colorimetric mode and Lateral Flow
Studio software (version 3.03.05, Qiagen). A reflectance signal intensity
for the test line of ≥50 mV was set as the cut-off value to identify
positive tests.
Ö. Akineden, et al. Food Control 109 (2020) 106952

Table 1
Cronobacter spp. (n = 41) strains used for inclusivity testing of the NALFIA.
Species Strain Origin Serotypea Biotypeb MLST Sequence typec

C. sakazakii (n = 22) DSM 4485T Type strain, clinical isolate, USA SO1 – ST8
DB-372/2k-06 PIF (≥4 months)1, Germany SO4 1 ST108
DB-382-3D3-06 PIF (≥4 months), Germany SO3 2 ST33
DB-376/2e-06 PIF (day 1), Germany SO1 2 ST108
DB-258-1-05 PIF (day 1), Germany SO1 1 ST4
DB-M2 PIF (≥6 months), Germany SO2 8 ST64
Db-246-3-05 PIF (≥6 months), Germany SO1 2 ST1
DB-277-1-05 PIF (≥6 months), Germany SO2 1 ST263
(DB)38a PIF (≥4 months), Germany SO4 1 ST248
DB-144a-5-05 PIF (≥4 months), Germany SO2 1 ST20
DB-73a/2-05 Whey powder, Germany SO3 1 ST4
DB-264-3-04 PIF (≥4 months), Germany SO1 1 ST40
DB-141a-1-05 PIF (≥4 months), Germany SO7 1 ST83
DB-280-2-05 PIF (≥6 months), Germany SO2 1 ST23
DB-185c/3-05 PIF (≥6 months), Germany SO7 1 –
DB-238/2-05 PIF (≥6 months), Germany SO4 1 –
DB-255/1-05 PIF (≥4 months), Germany SO2 1 –
DB-262/1-05 PIF (≥4 months), Germany SO2 1 –
DB-275/1-05 PIF (≥4 months), Germany SO3 1 ST20
DB-321/2c-06 PIF (≥4 months), Germany SO7 1 ST1
DB-338/3j-06 PIF (≥4 months), Germany SO1 1 ST1
DB-367/1c-05 PIF (≥4 months), Germany SO3 2 ST1
C. malonaticus (n = 8) DSM 18702T Type strain, clinical isolate, USA MaO2 9 ST7
DB-160a-6-05 Milk powder, Germany MaO1 9 ST211
DB-299-6y-06 PIF (≥4 months), Germany MaO2 9 ST7
DB-84a/1-05 PIF (≥8 months), Germany MaO1 5 ST60
DB-271-3-05 PIF (≥4 months), Germany MaO1 9 ST441
IB-37a-2003 PIF, Indonesia – 5 ST138
DB-143c-7-05 PIF (≥4 months), Germany MaO1 5 –
N30 Dried pasta, Japan – 9 ST291
C. turicensis (n = 4) DSM 18703T Type strain, clinical isolate (Switzerland) Ctur O1 – ST19
N35 Dried pasta, Germany – 16 ST251
N160 Dried pasta, Germany – 16 ST293
C32 Ready to eat salad, Germany – 16 ST252
C. muytjensii (n = 2) DSM 21870T Type strain, USA CmuytO2 – ST81
N40 Dried pasta, Germany – 15 –
C. dublinensis subsp. dublinensis DSM 18705T Type strain, environment, Ireland CdubO1 – ST106
C. dublinensis subsp. lausannensis DSM 18706T Type strain, water, Switzerland CdubO2 – ST80
C. dublinensis subsp. lactaridi DSM 18707T Type strain, environment, Zimbabwe CdubO1 – ST79
C. condimenti LMG 26250T Type strain, food, Slovakia CuniO1 – ST98
C. universalis NCTC9529 T Type strain, water, United Kingdom CuniO1 – ST54

1
recommended age for feeding.
T
Type strain; ATCC (American Type Culture Collection); DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Leibniz-Institute DSMZ); LMG
(Laboratorium voor Microbiologie, Gent, Belgien); NCTC (National Collection of Type Cultures, Culture Collection of Public Health England).
a
by PCR according to Sun et al. (2011).
b
according to Farmer (1980) and Iversen, Waddington, Farmer, and Forsythe (2006).
c
according to Baldwin et al. (2009); -, not defined.

2.6. Detection limit and specificity of the NALFIA
The test sensitivity (limit of detection, LOD) of the NALFIA system was
assessed using two type strains, C. sakazakii DSM 4485T and C. malonaticus
DSM 18702T, employing BPW and PIF as the sample matrix. The absence
of Cronobacter spp. in the PIF material was first confirmed by analysis
using method ISO/TS 22964:2006. A single colony of each strain from
Tryptic Soy Agar (TSA) was suspended in 10 ml of Tryptic Soy Broth (TSB)
and cultured at 37 °C for 18 h, the resulting bacteria density typically was
around 108 cfu/ml. Then, serial log10 dilutions (10−1 - 10−8) of each
culture were prepared in BPW or in reconstituted (10 g PIF plus 80 ml
BPW) PIF solution. For colony count, 2 × 100 μl of each dilution were
plated in duplicate onto Tryptic Soy Agar (TSA) and incubated overnight
at 37 °C. For preparation of artificially contaminated sample matrix, 1 ml
each of the 10−1 - 10−8 dilutions were added to test tubes containing BPW
(9 ml) or reconstituted PIF (9 ml) under gentle mixing, assuming that this
would yield final bacteria numbers of about 107 - 100 cfu/ml. The exact
bacterial concentration was obtained retrospectively from colony count
data. DNA was extracted from BPW or reconstituted PIF after incubation
by mixing 100 μl of sample with 400 μl of lysis buffer (RIDA gene, r-Bio-
pharm, Germany). The mixture was incubated at 95 °C for 5 min
(ThermoMixer, Eppendorf, Germany). The DNA concentration was de-
termined photometrically (NanoDrop One, Thermo Scientific, Germany),
then 5 μl of the mixture were used for PCR. Blank solutions of BPW or
reconstituted PIF were used as negative controls. Three independent re-
plicate analyses were performed for each series of experiments.
The reactivity pattern of the NALFIA was evaluated against a panel of
Cronobacter and non-Cronobacter strains grown from single colony iso-
lates. Inclusivity was studied by testing culture material of 22 C. sakazakii
and 8 C. malonaticus strains of various serotypes, biotypes and MLST
types (Table 1). The exclusivity was assessed with a panel of 11 strains
belonging to other Cronobacter species and 21 other non-Cronobacter
bacteria with relevance for PIF (Table 2). All bacterial strains were cul-
tivated on TSA at 37 °C for 24 h. DNA extraction from 2 to 3 colonies of
each isolate, suspended with 0.5 ml of lysis buffer in a 2 ml sterile mi-
crocentrifuge tube, was performed as described above. At least two in-
dependent DNA extractions and analyses were performed for each strain.
2.7. Detection of C. sakazakii and C. malonaticus in artificially
contaminated PIF
The applicability of the NALFIA system was evaluated using a

3
Ö. Akineden, et al. Food Control 109 (2020) 106952

Table 2
Non-Cronobacter (n = 21) strains used for exclusivity testing of the NALFIA.
Species Strain Origin/Source

Escherichia coli DSM 1103 (ATCC 25922) Clinic

Enterobacter cloacae subsp. cloacae DSM 30054T (ATCC 13047T) Type strain, clinic
Enterobacter cloacae subsp. dissolvens DSM 16657T (ATCC 23373T) Type strain, corn
Enterobacter aerogenes DSM 30053T (ATCC 13048T) Type strain, clinic
Enterobacter amnigenus DSM 4486T (ATCC 33072T) Type strain, environment
Klebsiella pneumoniae subsp. pneumoniae DSM 30104T (ATCC 13883T) Type strain
Klebsiella pneumoniae subsp. ozaenae DSM 16358T (ATCC 11296T) Type strain, clinic
Klebsiella oxytoca DSM 5175T (ATCC 13182T) Type strain, clinic
Yersinia enterocolitica subsp. palearctica DSM 11502 Clinic
Citrobacter freundii DSM 30039T (ATCC 8090T) Type strain
Hafnia alvei Sal3/2a-99 (2/14) Mincet meat
Pantoea agglomerans DSM 3493T (ATCC 27155T) Type strain, clinic
Proteus mirabilis DSM 788 (ATCC 14153) –
Proteus vulgaris DSM 30119 Faeces
Serratia marcescens 2/53 Meat
Shigela sonneii DSM 5570T (ATCC 29930T) Type strain
Salmonella enterica subsp. enterica DSM 10062 (ATCC 43845) Serovar Senftenberg
Pseudomonas aeruginosa DSM 939 (ATCC 15442) Environment
Pseudomonas putida F91-D Cream cheese
Acinetobacter baumannii DB-207a/3-05 PIF
Staphylococcus aureus DSM 20372 (ATCC 12598) Clinic, Enterotoxinogenic (seg, sei)

T
Type strain; ATCC (American Type Culture Collection); DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Leibniz-Institute
DSMZ); - not defined.

Fig. 1. Schematic representation of the principle of NALFIA with integrated DNA probe degradation.
Panel A: Presence of target sequence allows annealing of the primers and binding of the DNA probe labelled with digoxigenin and biotin to the DNA template.
Panel B: DNA probe is partially degraded by exonuclease activity of DNA polymerase, Panels C and D: Reactions on the NALFIA lateral flow strip. In negative tests (C)
intact DNA probe is captured by gold-labelled anti
-digoxigenin monoclonal antibody, this complex is then captured by streptavidin on the control line. For positive tests (D), some digoxigenin is released from the
probe and is then captured by gold-labelled anti
-digoxigenin monoclonal antibody. This complex passes the control line and is then immobilized at the test line (coated with goat anti mouse-antibody). . (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4
Ö. Akineden, et al.
Table 3
Oligonucleotide primer sequences for amplification of the rpoB gene.
Primer/probe Primer sequence
CsakFmm 5′-ACG CCA AGC CTA TCT CCA CG-3′
rpoB-RVmm 5′-AGA CCG CCC GGG CCA AGA TCA-3′
rpoB-SNDn 5′-DIG-GTGAAAGAGTTCTTCGGTTCAAGCCAGCTTTC-Biotin-3′
commercial product of PIF (recommended use for newborns) purchased
in a local German retail market. The absence of Cronobacter spp. in this
material was first confirmed by analysis using method ISO/TS
22964:2006. For artificial contamination, test portions of each 10 g of
this material were reconstituted with 89 ml of BPW immediately before
use. Contamination experiments were done with overnight cultures of
four different strains, C. sakazakii DSM 4485T, C. sakazakii DB84a-05, C.
malonaticus DSM 18702T, and C. malonaticus DB185-05, all in TSB.
Colony count and preparation of log10 dilution series were done as
described in 2.6. The 10−7 dilution was used for artificial contamina-
tion, based on the assumption that this dilution typically contained
about 101 - 102 cfu/ml. One ml of this dilution was added to recon-
stituted PIF, resulting in an estimated contamination level with Cro-
nobacter cells of 100-101 cfu per gram PIF. PIF (10 g) in blank BPW
(90 ml) was used as a negative control. Artificially contaminated sam-
ples were incubated at 37 °C for 24 h without agitation. Two portions of
each 100 μl of this non-selective enrichment broth were collected for
DNA extraction and analysis by NALFIA. Then, 0.1 ml the broth was
transferred into 10 ml selective enrichment broth (mLST/vancomycin
broth, ISO/TS 22964:2006 (IDF/RM 210: 2006) and incubated again at
37 °C for 18 h. Again, two 100 μl portions of this selective enrichment
broth were analysed by NALFIA. To quantitatively estimate the growth
of Cronobacter after selective enrichment, log10 serial dilutions (10−4 -
10−6) from the mLST/vancomycin broth were made with BPW, and
100 μl per dilution plated on Chromogenic Cronobacter Isolation (CCI)
agar (Oxoid). For each strain, the spiking experiment was repeated six
times.
3. Results and discussion
3.1. NALFIA test characterization and test performance
Combining the signal amplification power of PCR with the sensitivity
and simplicity of lateral flow immunoassays offers promising fields of
application in food safety. One major disadvantage of such rapid testing
methods is the risk of non-specific amplification, leading to false-positive
results. In the present study, a new NALFIA method, which is designed to
reduce false-positive results by employing a probe degradation step was
developed and applied for the detection of two important pathogens in
infant formula, C. sakazakii and C. malonaticus. Examples of lateral flow
devices obtained for negative and positive samples is shown in Fig. 2.
Instrumental evaluation of the test results by measuring reflectance en-
abled quantitative results which are more suitable for documentation
purposes. Overall, the test performance was found to be straightforward,
and the total test time was less than 2 h. NALFIA systems without in-
tegrated control via DNA probe degradation have been described for the
detection of some bacteria (Blažková et al., 2011; Seidel et al., 2017;
Zhang et al., 2017), and for parasites (Wu et al., 2017). To our best
knowledge, however, this is the first report that employs a hetero-
bifunctional DNA probe and detection of degradation for pathogens re-
lated to food safety, specifically for Cronobacter species.
The specificity of the NALFIA was determined using reference and field
strains covering all known Cronobacter species, plus a wide range of gram-
negative and gram-positive bacteria known to occur in the food environ-
ment (Tables 1 and 2). The NALFIA was highly specific for C. sakazakii and
C. malonaticus. The cross-reactivity testing of 41 Cronobacter and 21 non-
Cronobacter isolates revealed neither false positive nor false negative re-
sults (Table 4). The NALFIA assay targeted the rpoB sequence specific to C.
5
Food Control 109 (2020) 106952
Tm °C (nearest n)
60.36 °C
60.71 °C
65.07 °C
sakazakii and C. malonaticus, a chromosomal gene with a high level of
conservation (Mollet, Drancourt, & Raoult, 1997). The rpoB of both Cro-
nobacter species is suitable for detection with higher sensitivity and spe-
cificity than other target genes (Lehner, Fricker-Feer, & Stephan, 2012;
Stoop, Lehner, Iversen, Fanning, & Stephan, 2009). Previous studies have
shown that the rpoB gene is not suitable to clearly distinguish between
both C. sakazakii and C. malonaticus (Akineden et al., 2017). Since C. sa-
kazakii and C. malonaticus are by far the most commonly occurring Cro-
nobacter species in PIF, and to date are the only species related to PIF-
related infections of infants, the NALFIA appears to be primarily suitable
for monitoring of infant foods. Although it certainly cannot replace control
analyses by the reference method as required by European Commission
regulation 2073/2005 (European Commission, 2005), NALFIA could be
used as a supplementary rapid test for internal production control pur-
poses in PIF industry. The specificity pattern of the NALFIA described here
presents a complementary approach to a previous assay (Blažková et al.,
2011) for Cronobacter spp. which is based on the amplification of 16S
rRNA gene, in particular since the 16S rRNA gene does not allow the
unambiguous identification of Cronobacter spp. (Baldwin et al., 2009).
3.2. Limit of detection of the NALFIA
The sensitivity (limit of detection) of the NALFIA was evaluated by
using either BPW or Cronobacter-negative, reconstituted PIF, to which C.
sakazakii or C. malonaticus type strains had been added at defined cell
concentrations (101–107 cfu/ml). For these experiments, PCR amplifica-
tion was performed on samples after artificial contamination, without
prior enrichment. At an instrumental cut-off value of 50 reflectance units,
the detection limit for both Cronobacter species both in BPW and in PIF
was in a range of 103-104 cfu/ml in BPW (Fig. 3). The lateral flow reader
cut-off of ≥50 mV was defined because at this value, visual evaluation of
dipsticks also yielded consistently positive samples. Negative samples
consistently gave reflectance values of less than 5 mV after instrumental
reading. This test sensitivity was found to be highly reproducible, be-
cause the relative standard deviation (coefficients of variation of the
measurement signal, reflectance mV) were consistently less than 20% for
BPW and less than 25% for PIF in each three independent replicate
analyses for each strain and matrix. Visual evaluation of the NALFIA test
strips yielded identical sensitivity, Cronobacter at cell densities in a range
between 103 and 104 cfu/ml were clearly identified as positive in all
series of experiments. These results also confirm the test robustness with
regard to sample matrix interference. The detection limit of a previous
assay (Blažková et al., 2011) for Cronobacter spp. had been determined
for extracted DNA only, while in our study a thorough evaluation of the
detection limit of the complete analytical procedure was performed
based on bacteria numbers. Reconstituted infant formula was found to be
an ideal food substrate for NALFIA. Neither sample homogenisation nor
extensive sample cleanup was required for analysis. This enabled vir-
tually identical limits of detection for both Cronobacter spp. in BPW and
in PIF after 18 h of incubation at 37 °C. However, this may not be the case
if other food matrices had to be analysed.
As is reflected by results shown in Tables 4 and 5, the strongest
factor of variability was not related to assay repeatability but to strain
diversity. Depending on the specific isolate, the reflectance readings
varied substantially among 22 isolates of C. sakazakii (coefficient of
variation 76%) and C. malonaticus (39%), but all isolates were dis-
cerned as positive. However, the NALFIA has to be regarded as a qua-
litative rather than a quantitative assay.
Ö. Akineden, et al. Food Control 109 (2020) 106952

Fig. 2. Typical examples of NALFIA lateral flow test devices for positive and negative samples. A, negative result. B1, C. sakazakii DSM 4485T at approx. 106 cfu/ml.
B2, C. malonaticus DSM 17802T at approx. 106 cfu/ml.

Table 4
Specificity of the NALFIA.
Species Number of Strain diversity NALFIA result
strains
Serotypesa Biotypesb MLST typesc visual Reflectance mean ± standard deviation
(range, CV)

C. sakazakii 22 SO1, SO2, SO3, SO4, 1, 2, 8 ST1, ST4, ST8, ST20, ST23, ST33, all + 130 ± 99 (75–380, 76%)
SO7 ST40, ST64, ST83, ST108,
C. malonaticus 8 MaO1, MaO2 5, 9 ST7, ST60, ST138, ST211, ST291, all + 280 ± 110 (51–400, 39%)
ST441
C. turicensis 4 CturO1 16 ST19, ST251, ST252, ST293 all - all < 50
C. muytjensii 2 CmuytO2 15 ST81 all - all < 50
C. dublinensis subsp. dublinensis 1 CdubO1 n.d. ST106 – all < 50
C. dublinensis subsp. lausannensis 1 CdubO2 n.d. ST80 – all < 50
C. dublinensis subsp. lactaridi 1 CdubO1 n.d. ST79 – all < 50
C. condimenti 1 CuniO1 n.d. ST98 – all < 50
C. universalis 1 CuniO1 n.d. ST54 – all < 50
Other non-Cronobacter species 21 – n.d. – all - all < 50
(Table 2)

a
by PCR according to Sun et al. (2011); baccording to Farmer (1980) and Iversen et al. (2006); caccording to Baldwin et al. (2009); -, negative; +, positive; CV,
coefficient of variation.

NALFIA, 1) after non-selective enrichment in BPW and 2) after selective

Fig. 3. Characteristic dose-response curves of the NALFIA (instrumental reading,
peak height) for serial dilutions of type strains C. sakazakii DSM 4485T and C.
malonaticus DSM 17802T in either BPW or reconstituted PIF as the sample matrix.
Each data point represents the mean of three independent experiments. The mean
coefficients of variation for reflectance (peak height) of replicates at 104, 105, and
106 cfu/g in all series of experiments were 26%, 25%, and 32%, respectively.
3.3. Evaluation of the NALFIA as a means to obtain rapid results within the
enrichment procedure of the ISO/TS 22964:2006 reference method
In a last series of experiments, detection of C. sakazakii and C.
malonaticus in PIF at a low level (approximately 100-101 cfu/g) by
enrichment in mLST/Vancomycin broth, within the ISO/TS 22964:2006
reference method was studied. In each replicate experiment, the pre-
sence of Cronobacter was confirmed by completing the analysis by the
reference method, to verify that the artificial contamination had been
successful. In all six replicate experiments, NALFIA yielded highly po-
sitive instrumental reflectance readings for BPW samples (85–440 mV)
and for mLST/vancomycin samples (82–480 mV), and all replicate tests
were also clearly positive after visual evaluation (Table 5).
An inoculation level of approximately 1 cfu/g PIF was close to a
realistic contamination scenario of PIF, and still sufficiently high to
enable sufficiently accurate quantification. Natural contamination of
PIF produce often occurs at very low levels, usually a few cells per
100 g, as estimated for C. sakazakii (Osaili & Forsythe, 2009). Based on
re-analyses of colony counts of pre-enrichment BPW broth by micro-
biological plate count after incubation, the initial spiking level of 100-
101 cfu/g resulted in final counts of about 107 to 109 cfu/ml after 18 h.
Based on these findings, and considering the detection limit of 103-
104 cfu/g, the NALFIA appears to be capable to detect C. sakazakii and
C. malonaticus at initial levels of less than 1 cfu per gram of PIF. This
would compare well with previous reports which used cultural detec-
tion methods (Muytjens, Roelofswillemse, & Jaspar, 1988; Simmons,
Gelfand, Haas, Metts, & Ferguson, 1989), although this range was not
studied, because homogeneous artificial contamination of PIF at levels
much lower than 1 cfu per gram is difficult to achieve in practice. In any
case, the NALFIA is comparable with a similar test described by
Blažková et al. (2011), based on the 16S rRNA gene, who claimed a
detection limit of less than 10 cells/10 g of PIF. Although these authors

6
Ö. Akineden, et al.
Table 5
Comparison of NALFIA (visual and instrumental evaluation) with reference method ISO/TS 22964:2006 for PIF which was artificially contaminated with Cronobacter
strains at a low level (100-101 cfu/g). In each experiment, NALFIA was applied to non-selective enrichment broth (BPW) at 24 h, and to selective enrichment broth
(mLST/Vancomycin) at 48 h. For each strain, six independent replicate experiments were performed.
Strain Non-selective pre-enrichment in BPW
Reference method NALFIA visual NALFIA instrumental mean ± SD
(range)
C. sakazakii DSM 4485T + + 310 ± 43 (250–380)
C. sakazakii DB-185c/3-05 + + 229 ± 57 (120–290)
C. malonaticus DSM 18702T + + 240 ± 120 (150–440)
C. malonaticus DB-84a/1-05 + + 220 ± 97 (85–370)
+ positive results in all six replicates.
mentioned that their assay was successfully used to test PIF, experi-
mental data were not provided. In the present study, a comprehensive
evaluation of the NALFIA properties has been done, including enhanced
specificity controls and rigorous detectability checks with each two
strains of C. sakazakii and C. malonaticus, both in BPW and PIF, clearly
demonstrating the suitability and robustness of the assay. Furthermore,
quantitative measurement of dipsticks by an automated dipstick reader
device enabled an objective evaluation of the repeatability of the
NALFIA results for both species in real sample material (Table 5).
4. Conclusion
The newly developed NALFIA with integrated DNA probe de-
gradation enabled rapid (90 min) detection of the two most important
Cronobacter spp., C. sakazakii and C. malonaticus, in PIF after 18 h non-
selective pre-enrichment in BPW. The NALFIA was both sensitive and
highly robust towards sample matrix. Reconstituted infant formula was
found to be a convenient food matrix for NALFIA, test sensitivity in PIF
was equal to that obtained in BPW. Both visual and instrumental eva-
luation are possible, the latter offering the advantage of an easy doc-
umentation of the results. However, the NALFIA presents a significant
step towards testing in a laboratory-independent environment. Further
work therefore aims at simplifying the PCR amplification step, for ex-
ample by using battery-operated cycler based on convection, to enable
testing at the point of care within the dairy industry.
Funding
This work was supported in part by the Federal Ministry of
Education and Research (BMBF) of Germany (Food supply and analysis
(LEVERA), funding code 13N12611).
Declaration of competing interest
The authors declare no conflict of interest. T. Wittwer and K. Geister
are employees of R-Biopharm AG. This manuscript and the experi-
mental work described therein do not involve a commercial product.
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