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Food Control 40 (2014) 359e367

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Review

Rapid and standardized methods for detection of foodborne


pathogens and mycotoxins on fresh produce
F. Yeni a, b, S. Acar b, .G. Polat b, Y. Soyer b, H. Alpas b, *
a
b

Department of Earth System Sciences, Middle East Technical University, 06800 Ankara, Turkey
Department of Food Engineering, Middle East Technical University, 06800 Ankara, Turkey

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 14 June 2013
Received in revised form
2 December 2013
Accepted 17 December 2013

Due to the increase in consumption of fresh produce regarding to the health demand in the last decades,
a considerable portion of foodborne outbreaks has been trackbacked to contaminated fresh produce,
which have appeared as highly possible vehicles for foodborne outbreaks nowadays. Delays in detection
of pathogens and mycotoxins on fresh produce hindered the trace-back investigations in nding the
source and revealed the urgent need of rapid and reliable methods. In the frame of this review, we
summarized available fast, reliable and standardized methods (conventional, molecular, rapid and
recently developed methods) used for detection of the most common foodborne pathogens and mycotoxins which are the most likely causative agents of outbreaks caused by contaminated fresh produce.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Detection
Rapid methods
Foodborne pathogens
Mycotoxins
Fresh produce

Contents
1.
2.

3.

4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Conventional methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
2.1.
Sampling and isolation procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
2.2.
Detection procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
2.3.
Advantages & disadvantages of conventional detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Molecular methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
3.1.
Sampling and isolation procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
3.2.
Detection procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
3.3.
Advantages & disadvantages of molecular detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
Recently developed detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
4.1.
Sampling and isolation procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
4.2.
Detection procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
4.3.
Advantages & disadvantages of recently developed detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

1. Introduction

* Corresponding author. Tel.: 90 (312) 210 56 18; fax: 90 (312) 210 27 67.
E-mail address: imah@metu.edu.tr (H. Alpas).
0956-7135/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.12.020

In the last few decades, volume of international trade in food


items rapidly increased while whole agro-food markets has globalized which resulted in food safety problems due to different
safety practices of countries in all around the world. On the other
hand, since 1980s, consumption of fresh produce has been in an

360

F. Yeni et al. / Food Control 40 (2014) 359e367

upward trend due to the consumers demanding healthy food


(FAOSTAT, 2012; Huang, 2004). Moreover, this demand continues
through the year and compensated by greater export volume while
increasing the risk of contamination during prolonged duration of
storage and transportation stages (Lynch, Tauxe, & Hedberg, 2009).
This trend resulted in elevated numbers of foodborne outbreaks
caused by contaminated fresh produce in the recent years in all
around the world. For instance, in the period November 2010 November 2012; 5191 people were infected with foodborne pathogens on raw fresh produce items and consequently 95 people died
due to the outbreaks in European countries, USA, Canada and Japan
(CDC, 2013). According to data published by European Food Safety
Authority (EFSA), food of non-animal origin, which mainly
comprised of processed and non-processed fresh produce items, is
responsible for 10% of the total foodborne outbreaks, 26% of the
cases, 35% of the hospitalizations and 46% of the deaths between
2007 and 2011 in Europe (Member States of European Union,
Norway and Switzerland) (Cerroni et al., 2010). On the other hand,
5630 people infected, 859 people hospitalized and 11 people died
due to consumption of fresh produce including food items which
were contaminated with foodborne pathogens between 2006 and
2010 in USA (CDC, 2013). In terms of mycotoxins, although number
of outbreaks have declined as a result of strict limits set by national
and international regulatory agencies, there can be occasional
outbreaks due to inappropriate storage environmental conditions
in the underdeveloped countries (Adams & Moss, 2006). In the last
10 years, two outbreaks occurred Kenya and Brazil while 837
people were intoxicated and 157 people died because of mycotoxin
containment in cereals (Elizaquivel, Sanchez, & Aznar, 2012; Lima
et al., 2010). Also, in the USA, 70 people affected 41 people hospitalized and three people died due to in mushrooms and pineapple
containing mycotoxin between 2001 and 2010 (CDC, 2013).
In the light of these outbreak experiences, fresh produce has
been accepted as a highly possible vehicle for foodborne outbreaks
and routinely monitored for pathogen and mycotoxin contamination during the trace-back investigations (Lynch et al., 2009). The
trace-back investigations ascertained the prevalence of foodborne
pathogens and mycotoxins (e.g. as aatoxins, ochratoxin A, citrinin
and patulin) amongst the major sources of recent outbreaks. For
instance, Salmonella enterica, Staphylococcus aureus, pathogenic
Escherichia coli, Listeria monocytogenes, Clostridium spp., Shigella
spp. and Yersinia spp. are responsible for 42% of the total outbreaks
occurred while pathogenic E. coli species account for the great
majority of hospitalizations and death between 2007 and 2011
(Cerroni et al., 2010). These seven pathogens caused 99 outbreaks
via fresh produce items between 2006 and 2010 in USA (CDC, 2013).
On the other hand, although occurrence of outbreaks caused by
mycotoxin containment have been prevented almost completely
due to strict national and international regulations, mycotoxins still
pose a risk to food of plant origin (especially to nuts, seeds and
spices among fresh produce) via occasional outbreaks due to
inappropriate storage and environmental conditions (Adams &
Moss, 2006; Forsythe, 2010). Therefore, it is evident that efforts
to prevent all the outbreaks could not have been completely successful due to time-consuming process of available detection
methods used in routine screening practices.
Screening practices by microbiological testing formerly applied
only to nished products in order to prevent outbreaks, however,
modern surveillance systems aims to ensure food safety by preventing or minimizing contamination of food items along the food
chain beginning with the production of raw materials (Forsythe,
2010). Preventing contamination of fresh produce is of vital
importance in several ways. One critical point is that fresh produce
items are often consumed raw and not exposed to enough postharvest treatment or cooking to eliminate or reduce pathogens

before consumption (Rambo & Pillai, 2011; Ribot, Hyytia-Trees, &


Cooper, 2008). Moreover, as the produce items are essential raw
materials for food manufacturers (Carlin, 2007), potential serious
consequences of contaminated produce items in terms of mixed or
prepared food products should also be taken into account. Also,
contamination of the produce items in the production phase or in
early stages of postharvest handling will increase the risk of cross
contamination and spread of pathogens locally, nationally and
internationally due to wider range of distribution of contaminated
produce (Gorny, 2006; Hoofar et al., 2011). Recent outbreaks supported this argument as the trace-back investigations demonstrated that the produce items are often contaminated during the
production phase at a single produce company. On the other hand,
if contamination occurs, complete elimination of foodborne pathogens and mycotoxins from the produce is not always possible
(Bennett & Klich, 2003; Parish et al., 2003).
When contamination occurs in any stage of food supply chain
and it cannot be eliminated before the produce item becomes
available for consumption, detecting the contaminated products
appears as the best option in order to prevent any public health
issue. Because the fresh produce items have a short shelf life and
they are consumed instantly, sampling and detection of contaminated products must be considerably rapid and reliable. Many
outbreaks caused by contaminated fresh produce items reveal the
urgent need of using rapid methods in national and international
surveillance systems. More recently, due to the errors and delays in
detection of the source of the outbreak, the E.coli O104:H4 outbreak
in Europe 2011 became the biggest foodborne outbreak occurred in
Europe (Sprenger et al., 2011; WHO, 2011).
There are general standard methods for determination of the
foodborne pathogens and mycotoxins in food products including
conventional methods such as culture and microscopic methods,
chemical and biological methods (immunological, molecular genetic
methods, gel diffusion), there are also more rapid methods which
were recently developed including physical methods (biosensors,
impedance, microcalorimetry, ow cytometry, biosys instrument)
and bioassays (Jay, Loessner, & Golden, 2005). While antibody and
nucleic acid-based rapid assays prevailed over the methods which use
basic technologies during 1990s while real-time PCR (rtPCR), microarrays, and biosensors have emerged as recent developments in the
eld of pathogen testing market in the 2000s (Mattingly, Butman,
Plank, Durham, & Robison, 1988). Therefore, current surveillance
systems have been changed from culture-conrmed microscopic
methods to rapid and commercial non-culture methods in order to
prevent foodborne diseases (Jones & Gerner-Smidt, 2012). However,
all of the recently emerged methods can not be applied to fresh produce and the available methods used for determination and
enumeration of pathogens on raw produce are generally modications of the ones developed and validated for processed food products
of plant origin (Beuchat, 2006).
As a natural consequence of the problems mentioned above,
using rapid and reliable methods is of vital importance in the surveillance studies in order to prevent the outbreaks, to detect these
organisms in the early warning and notication systems and to
trace-back the source pathogens. In the context of this paper we
have reviewed the conventional analytical methods, which are
widely used to detect foodborne pathogens and mycotoxins on
fresh produce as well as more rapid methods including validated
rapid commercial protocols and developments in the available
molecular and emerging methods.
2. Conventional methods
Validation is essential for standardization of a method as well as
extent its usage in all around the world. Many national and some

F. Yeni et al. / Food Control 40 (2014) 359e367

international institutions validate developed methods due to the


sensitivity and specicity of the method which are approximately
at the level of 95% concerning the methods available for foodborne
pathogens (Forsythe, 2010). Methods for detection of foodborne
pathogens and mycotoxins on fresh produce validated by International Organization for Standardization (ISO) are used with minor
adaptations in the food control and reference laboratories of almost
every country. However, some countries develop their own
methods or revalidates available methods according to their own
legal requirements. For instance, European Union (EU) countries
prefer to use the protocols, which are modied from ISO methods
by European Committee for Standardization (CEN), on the other
hand Bacteriological Analytical Manual of United States Food and
Drug Administration (FDA-BAM) is used as a guideline in public
health laboratories in the USA. Conventional methods validated by
these institutions are listed in Table 1.
The conventional methods listed in Table 1 are composed of
culture-based methods for detection and enumeration of foodborne pathogens and high-pressure liquid chromatography (HPLC)
based methods are used for determination and identication of
mycotoxins. Among the conventional methods validated by FDA,
CEN and ISO, solely culture-based methods are available for Salmonella, Yersinia enterocolitica, L. monocytogenes, Clostridium perfringens, and coagulase-positive staphylococci including S. aureus.
Moreover, some other molecular techniques are also available for
detection of foodborne pathogens together with culture-based
methods. For instance, real-time PCR is used for determination of
E. coli (STEC) O157, O111, O26, O103 and O145 serogroups in the
methods validated by CEN/ISO and DNA hybridization technique is
used for detection and enumeration of Shigella in the method
validated by FDA.
In terms of biological toxins, immunological, molecular and
chromatographic techniques form the basis of validated methods.
Concerning Staphylococcal enterotoxins; toxins are extracted via
chromatographic techniques, detected and identied via enzymelinked immunosorbent assay (ELISA) and Enzyme-linked brinolytic assay (ELFA) techniques according to the methods approved by
FDA. Likewise, botulinum toxins are detected via ELISA technique
and mouse bioassay and groups of toxins identied via PCR according to the methods approved by FDA.
On the other side, HPLC appears as the basic technique for
determination of mycotoxins among the conventional methods
available for fresh produce. All of the methods validated by CEN/ISO
and FDA are based on this technique with minor differences such as
with immunoafnity column clean-up and post-column devrivatization, or uorescence detection.
2.1. Sampling and isolation procedures
In a routine sampling process for fresh produce items possibly
contaminated with pathogens, solid fresh produce items are
transported to laboratory rapidly in chilled or frozen state in order
to prevent growth and death of microorganisms and then generally
25 g of sample is taken with aseptic tools and diluted to 1:10
dilution (Stewart & Gendel, 1998). After sampling, there are some
basic steps to isolate the target organism from the food namely,
homogenization for solid samples, pre-enrichment for recovery of
injured cells, enrichment for suppression of non-target organisms,
plating with selective, non-selective or semi- selective agars for
distinguishing target pathogen and ensuring the purity of the isolates, respectively (Forsythe, 2010). Enrichment steps for pathogens
and extraction and concentration steps for toxins in food samples is
essential prior to identication of pathogens and toxins in order to
meet the sensitivity levels of available detection methods
(Mattingly et al., 1988).

361

Sampling of food items for analysis of mycotoxinsis considerably


different from the strategies applied for foodborne pathogens.
Sampling step is the main reason of erroneous results along the
detection procedure of mycotoxins due to inhomogeneousdistribution of mycotoxins and highly contaminated spots in lots of
food items.Therefore quantication of mycotoxin containment of
food productsgenerally can not be calculated with the exact certainty (Reiter, Zentek, & Razzazi, 2009). However, sampling procedures of mycotoxin producing fungi are performed almost in the
manner with the protocols applied for bacterial foodborne
pathogens.
Following the sampling step, mycotoxins are extracted from the
food item via organic solvents and interfering substances are
removed in the clean-up step in chromatographic techniques
(Reiter et al., 2009).After the clean-up step, high performance liquid
chromatography (HPLC) thin layer chromatography (TLC) is used as
conventional methods in order to quantify the mycotoxin
containment in fresh produce items.
Although the above mentioned procedure is simple, food scientists face with more complicated problems because of inherent
properties of fresh produce samples. In addition to differences in
size, shape and surface morphology of fresh produce precluding the
possibility of applying a single standardized procedure for sampling in order to detect and enumerate foodborne pathogens, there
are also difculties associated with homogenized, blended or
macerated tissues during preparation of the samples such as the
inhibitory effects of organic acids in many fruits or some other
antimicrobial compounds naturally available in tissues of vegetables, herbs and spices (Beuchat, 2006; Mattingly et al., 1988).
Moreover, because fresh produce is often consumed as mixed
food products such as salads or garnish, determining the contaminated source is a challenge for investigators in the situation of a
fresh produce related outbreak (Berger et al., 2010). However,
sampling stage during the trace-back investigations of an outbreak
is relatively less complicated because there is often a suspected
food product. On the other hand, food items are randomly sampled
in all stages of a food supply chain during routine screening practices according to the ultimate goal of modern surveillance systems.
However, the sample may not be representative of the food analyzedin random sampling because of the presence of different
species in a sample and homogeneity of the solid food samples
(Forsythe, 2010).
2.2. Detection procedures
Subsequent to isolation steps, mainly biochemical identication
tests are used for detection of foodborne pathogens in conventional
methods (Forsythe, 2010). Among the culture-based conventional
methods, standard plate count is the longest available detection
and enumeration method (Fung, 2006). Although this method is
simple and widely used for decades, it requires a large amount of
laboratory equipment, labor and time. As an alternative to standard
plate count, more rapid and simpler culture-based enumeration
methods introduced such as viable cell counts, differential counts,
pathogen counts and some commercial methods including spiral
plating, the isogrid system, petrilm method, redigel system in the
last 25 years (Fung, 2006). Also, incorporation of chromogenic and
uorogenic substrates into culture media can be regarded as recent
developments in the biochemical identication of pathogenic microorganisms (Mattingly et al., 1988).
In terms of mycotoxins, there are many protocols based on
chromatographic techniques, mass spectrometry, immunological
methods and biosensors applied for detection of mycotoxins in
cereals and diary products (Maragos & Busman, 2010; Prieto-Simon
& Campas, 2009; Reiter et al., 2009; Shephard et al., 2012), however,

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F. Yeni et al. / Food Control 40 (2014) 359e367

Table 1
Standardized conventional methods for detection of foodborne pathogens and mycotoxins on fresh produce.
Pathogen

FDA/BAM

CEN

ISO

Salmonella

Chapter 5, 2011
Culture-based horizontal
method for detection and enumeration

E. coli

Chapter 4, 2002
Chapter 4A, 2011
Culture-based horizontal
methods for detection and
enumeration of pathogenic
E. coli except EHEC of serotype
O157:H7

EN ISO 6579:2002
EN ISO 6579:2002/AC:2006
EN ISO/TS 6579-2:2012
Culture-based horizontal method for detection
and enumeration
CEN ISO/TS 13136:2012
rtPCR-based horizontal method for detection
of E. coli (STEC) and
O157, O111, O26, O103 and
O145 serogroups

EN ISO 16654:2001
Culture-based horizontal method
for detection
EN ISO 21567:2004
Culture-based horizontal method
for detection

ISO 6579:2002
ISO/NP 6579-1
ISO/PRF TS 6579-2
Culture-based horizontal
methods for detection, enumeration
ISO/PRF TS 13136
rtPCR-based horizontal method for
detection of E. coli (STEC) and
O157, O111, O26, O103 and O145 serogroups
ISO 7251:2005
Culture-based horizontal method
for detection and enumeration
ISO 16654:2001
Culture-based horizontal
method for detection
ISO 21567:2004
Culture-based horizontal method
for detection

EN ISO 10273:2003
Culture-based horizontal method
for detection

ISO 10273:2003
Culture-based horizontal
method for detection

EN ISO 11290-1:1996
EN ISO 11290-2:1998
EN ISO 11290-1:1996/A1:2004
EN ISO 11290-2:1998/A1:2004
Culture-based horizontal methods for
detection and enumeration
EN ISO 6888-3:2003
EN ISO 6888-3:2003/AC:2005
Culture-based horizontal method for enumeration
and detection of
coagulase-positive staphylococci
including S. aureus

ISO 11290-1:1996
ISO 11290-2:1998
Culture-based horizontal method
for detection and enumeration

EN ISO 7937:2004
Culture-based horizontal
method for detection and enumeration

ISO 7937:2004
Culture-based horizontal
method for detection and enumeration

EN 14123:2007
HPLC method with immunoafnity
column clean-up (IAC) and post-column
derivatization (PCD) for determination of
aatoxin B1 and the sum of aatoxin
B1, B2, G1 and G2 in hazelnuts,
peanuts, pistachios, gs
EN ISO 16050:2011
Reverse-phase HPLC method with
IAC and PCD for determination of
aatoxin B1 and the sum of aatoxin
B1, B2, G1 and G2 in cereals, nuts,
and derived products
EN 15829:2010
HPLC method with IAC and FD
for determination in currants,
raisins, sultanas, mixed dried
fruit and dried gs

ISO 16050:2003
Reverse-phase HPLC method
with IAC and PCD for determination
of aatoxin B1 and the sum of
aatoxin B1, B2, G1 and G2 in
cereals, nuts, and derived products

E. coli O157

Shigella

Y. enterocolitica

L. monocytogenes

Staphylococcus

C. perfringens

C. botulinum

Aatoxin

Ochratoxin A

Chapter 6, 2001
Culture-based horizontal
method &DNA hybridization
method for detection and
enumeration
Chapter 8, 2007
Culture-based horizontal
method for detection and
enumeration
Chapter 10, 2011
Culture-based horizontal
method for detection and
enumeration

Chapter 12, 2001


Culture-based horizontal
method for detection and
enumeration of S. aureus
and/or its enterotoxins
Chapter 13A, 2011
Extraction of enterotoxins
by chromatographic techniques &
culture-based enumeration
method & commercial test kits
using immunoenzymatic techniques
for detection and identication of
staphylococcal enterotoxins
Chapter 16, 2001
Culture-based horizontal method for
detection and enumeration of
C. perfringens and/or its enterotoxins
Chapter 17, 2001
Mouse bioassay or ELISA-based
technique for detection of botulinum
toxins & pcr- based method for
identication of C. botulinum
Chapter 18, 2001
TLC or HPLC method for
determination of aatoxins

Chapter 18, 2001


TLC or HPLC method for
determination of ochratoxin

ISO 6888-3:2003
Culture-based horizontal
method for enumeration
and detection of coagulase-positive
staphylococci

F. Yeni et al. / Food Control 40 (2014) 359e367

the only conventional methods available for detection of mycotoxin


containment in fresh produce are thin layer chromatography (TLC)
and high performance liquid chromatography.
2.3. Advantages & disadvantages of conventional detection
methods
Culture-based methods and chromatographic methods constitute the large majority of the conventional methods, which were
validated and standardized for international use for detection of
foodborne pathogens and mycotoxins. The culture-based conventional methods are the basic tools used for detection of foodborne
pathogens in all around the world for their reliability in efciency,
sensitivity to target organism, and application to a wide range of
food matrices (e.g. all food ad feed stuff) and environments of food
production and handling whereas more rapid protocols based on
recent developments in food science can be applicable for a limited
food matrix.Moreover, conventional methods are still regarded as a
more reliable option for conformation of the obtained resultsin
emergency situations. For instance,the false-negative results of
rapid methods may cause expansion of the outbreak when there is
a suspicion of contamination, or the false-positive results of rapid
methods may cause a delay in nding the real source when there is
an ongoing epidemiological investigation of an foodborne outbreak
(Hoofar et al., 2011).
Beyond these advantages, culture-based conventional isolation
and detection methods require a considerable amount of laboratory
equipment, large amounts of medium, and several days to detect
the target pathogens mainly because of multiple enrichment steps.
In addition to these disadvantages,laboratory personal should be
trained to prepare the samples and interpret the results of the
culture-based conventional methods (Forsythe, 2010; Stewart &
Gendel, 1998). Likewise, HPLC and TLC based methods requires
considerable amount of time, labor and instrumentation cost
although these techniques are widely used in all around the world
due to their high sensitivity and reliability (Prieto-Simon & Campas,
2009).
Therefore, more rapid and reliable methods such as molecular
and immunological methods and bioassays are needed to be
routinely used for screening practices of foodborne pathogens and
mycotoxins in order to prevent public health issues by considering
the speed and amount of transnational movement of fresh produce
items in the modern agro-food markets.
3. Molecular methods
Emerging pathogens, which are not detectable by conventional
microbiological methods, are growing in importance and time
plays a large role in industrial processes (Lofstrom, Krause, Josefsen,
Hansen, & Hoorfar, 2009). As a result of that; there is an increase of
nucleic acid (DNA and RNA)-based assays for the differentiation and
identication of foodborne pathogens. Due to rapid and reliable
detections and differentiations of foodborne pathogens, DNA
methods including polymerase chain reaction (PCR), pulsed-eld
gel electrophoresis, ribotyping, plasmid typing, randomly amplied polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) are generally used. There are some automated
versions of these methods and some can be applied in kits that
enable to recover pure DNA. PCR- based methods have beenthe
widely known and used method among them.
3.1. Sampling and isolation procedures
DNA isolation from food samples is a crucial step in all molecular
detection methods. First, the sample should be kept in an

363

appropriate temperature for a determined time period. After storage, the sample is extracted in a buffer system and then the supernatant is usually treated with proteinase K and CTAB solution
(headecyltrimethylammonium bromide with NaCl). DNA is
precipitated, the purity of DNA is checked, which is done generally
by gel electrophoresis methods (Naravaneni & Jamil, 2005). Lastly,
the extracted DNA is used for PCR and other molecular methods.
For DNA extraction, there are also commercially available kits such
as QIAamp DNA Stool Mini kit (Qiagen) (Fukushima, Tsunomori, &
Seki, 2003). After DNA extraction, the procedure specic to
method is applied depending on the type of molecular technique.
3.2. Detection procedures
PCR is a highly effective application that uses enzymes to
amplify a single colony of DNA by 106-fold in a few hours. It can
differentiate single-nucleotide polymorphisms (SNPs) and also can
be designed as multiplex tests. Thus it is able to detect several
pathogens depending on specic DNA regions of organisms.
Recently, it has been used to detect foodborne bacterial pathogens
such as viable E. coli O157:H7, Salmonella and L. monocytogenes cells
in fresh-cut vegetables (Elizaquivel et al., 2012).
Real-time PCR (rtPCR) differs from conventional PCR in various
ways; such as results are obtained (i) in real-time thus there is no
need of gel-based detection, (ii) in a very short time since the
amplication cycles are shorter and (iii) by using many different
kind of heating equipment, illumination source and detectors
(Kubista & Zoric, 2005). SYBR Green (a cyanine dye) I assay is the
most economic and easiest one among other PCR assays. SYBR
Green I is a uorescent dye to which double stranded DNA binds
specically. When the amount of double stranded amplicon increases, the intensity of the SYBR Green I uorescence increases
and it is monitored by rtPCR throughout amplication. In 2003, 17
species of foodborne and waterborne pathogens (enteroinvasive E.
coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, Salmonella spp., Shigella
spp., Y. enterocolitica, Yersinia pseudotuberculosis, Campylobacter
jejuni, Vibrio cholera, Vibrio parahaemolyticus, Vibrio vulnicus,
Aeromonas spp., S. aureus, C. perfringens, and Bacillus cereus) in
stools are tested by SYBR Green Light Cycler PCR by identifying their
melting temperatures (Fukushima et al., 2003). At the end, the
detection limit was calculated to be 105 cells/g, but there was a
need of overnight enrichment. Another SYBR Green assay is performed to detect Salmonella in alfalfa sprouts, milk and ground beef
with the combination of IMS (immunomagnetic separation) before
the PCR and detection degree was 1.5 cells/26 g of sample in 13 h
(Mercanoglu & Grifths, 2005). Since the identication of amplicons is based on only primers and melting temperature; sequencespecicity cannot be detected. Thus a combination with specic
probes is usually required to get over the problems of mispriming
and genetic variations. Another illumination source is the TaqMan
process that uses sequence-specic probe with a uorescent reporter and quencher dye. Firstly the probe binds to a specic
sequence and then Taq polymerase cleaves the bound by separating
the reporter from quencher during amplication proceeds. The
intensity of the uorescence, which changes according to the
number of amplicons, is measured. A TaqMan quantitative realtime PCR is developed to detect only live Salmonella cells and the
improved rtPCR was found to give fast and accurate results for
viable Salmonella detection for spinach, tomatoes, jalapeno and
serrano peppers (Gonzalez-Escalona et al., 2009). The other rtPCR
assay uses molecular beacon technique in which sequence-specic
probes having hair-pin or stem-loop oligonucleotide are used.
When the probes -consisting of a uorophore and a quencherhybridizes to a specic target, reporter separates from the quencher

364

F. Yeni et al. / Food Control 40 (2014) 359e367

and uorescence intensity increases. Molecular beacon-PCR is


studied to detect the presence of Salmonella spp. with fresh-cut
produce such as cantaloupe, mixed-salad, cilantro, and alfalfa
sprouts and as a result, 1e4 cfu/PCR reaction could be detected
(Liming & Bhagwat, 2004). A similar result was obtained in a
L. monocytogenes study that is performed on articially inoculated
fresh-cut produce such as cantaloupe and mixed-salad. At the end,
4 to 7 cfu/25 g of articially contaminated produce could be
detected (Liming, Zhang, Meng, & Bhagwat, 2004). Fluorescence
resonance energy transfer (FRET) rtPCR which uses energy transfer
between the donor and the acceptor molecules has also been
studied for detection of pathogens in food samples. With the help
of high-throughput automated DNA extraction, 32 food specimens
were processed and assayed in less than 2 hours and as a result, a
lower limit of 1.5  102 and 1.5  105 CFU/mL without preenrichment was achieved (Olsen, Gibbins, & Grayson, 2009).
Multiplex PCR, on the other hand, is the simultaneous amplication of more than one target sequence in a single reaction
(Henegariu, Heerema, Dlouhy, Vance, & Vogt, 1997). Differently
from individual rtPCR applications, fewer reactions are run, thus it
conserves the expensive reagents such as dNTPs, enzymes, primers,
etc. Therefore, at the end, the economic value of multiplex PCR
becomes lower than individual PCR. When sample amount is
limited, multiplexing permits more targets to be analyzed using a
single aliquot of sample material, thus it preserves limited amount
samples that should be tested for detection of several pathogens. In
a different manner from individual PCR methods, multiplex PCR
uses data, which has an improved quality, since the target of interest is normalized to the endogenous control within the same
aliquot of the sample, which at the end, allows for increased reliability. But, to use a successful multiplex PCR, one should be careful
about: relative concentration of primers, PCR buffer concentration,
balance between magnesium chloride and deoxynucleotide concentrations, cycling temperatures and amounts of template DNA
and Taq DNA polymerase (Markoulatos, Siafakas, & Moncany,
2002). The sensitivity of multiplex PCR is studied on articially
inoculated cleaned and packed spinach and lettuce and it was
observed that it was able to detect 0.9, 1.8 and 4.8 CFU/reaction of
E. coli O157:H7, Salmonella, and S. aureus, respectively, corresponding to 103 CFU g1 each (Elizaquivel & Aznar, 2008).
Nucleic acid sequence based amplication (NASBA) differs from
PCR application as there is no need of a thermal cycler and also it is
performed in isothermal conditions (Compton, 1991). It is usually
carried out to amplify RNA and cDNA (Lauri & Mariani, 2009).
NASBA has the capability to detect viable microorganisms in
different kind of samples including environmental and food
matrices (Chan & Fox, 1999; Cook, 2003).
The BAX system (DuPont, Qualicon, Wilmington, DE), a commercial polymerase chain reaction-based instrumentation, provides processes up to 96 unique samples within four hours after
sample preparation (Bailey, 1998; Silbernagel, Jechorek, Carver,
Barbour, & Mrozinski, 2003). And the results are useable as soon
as the following day and are distinctly displayed on screen with a
simple positive or negative report. It is available to be used for
screening Salmonella, E. coli O157:H7, L. monocytogenes, etc.
(Bhagwat, 2003; Stewart & Gendel, 1998). Sample preparations are
based on the standard protocols for each food type. Samples are
then heated in a lysis reagent solution to separate the bacterial cell
wall and release the DNA. PCR tablets, which contain all the reagents necessary for PCR plus uorescent dye, are hydrated with
lysed sample and processed in the cycler/detector. Within a few
hours, the PCR amplies a DNA fragment that is specic to the
target. The amplied DNA generates a uorescent signal, which the
BAX system uses to analyze the results which are then displayed
as simple positive or negative symbols (Becker, Jordan, & Holzapfel,

2005). BAX system was studied with retail sprouts and mushrooms
for contamination with E. coli O157:H7, Salmonella, Listeria spp., and
L. monocytogenes in 2003. Failure of the method is measured as 0.7%
and thus reagent failures and the inhibition of PCR by plant compounds were dened as uncommon. It was observed that the
sensitivity of the test is not consistent among food types and microorganisms. Thus, it was concluded that test sensitivity is affected
by the type of produce involved and is probably related to the
growth of pathogens in the resuscitation and enrichment media
(Strapp, Shearer, & Joerger, 2003).
3.3. Advantages & disadvantages of molecular detection methods
Molecular methods allow for sensitive and rapid detection of
several pathogens. The results can be obtained in shorter times as
in a day such as in real-time multiplex PCR (de Boer, Ott, Kesztyus, &
Kooistra-Smid, 2010). The sensitivity of a molecular method can be
set and improved by designing new primers, probes, using optimum conditions etc. thus, these methods are available to any
changes regarding to the requirements of detection level of pathogens (Yang et al., 2002). Also, the molecular methods give reliable,
high-throughput, reproducible and specic results (Klein, 2002;
Lindstrom et al., 2001). In addition, species identication, quantication and subtyping can be performed together with species
detection during molecular methods, so they allow for further data
on the phylogenetic characteristics of the strains identied
(Amagliani, Omiccioli, Brandi, Bruce, & Magnani, 2010; Girones
et al., 2010).
The positive result obtained a molecular method can be only
regarded as presumptive and must be conrmed by standard
methods. Thus, a molecular method alone cannot be used to detect
the source of a foodborne outbreak or identify the pathogen. Also,
the most rapid methods lack of sufcient sensitivity and specicity
for director testing, foods still need to be culture-enriched before
analysis. In addition, molecular methods are food and microorganism dependent. Besides, these methods can be used to detect
cell, but cannot be used to detect the toxin occurrence.
Despite the advantages of PCR such as being sensitive,
commercially available; PCR applications have some drawbacks. For
instance, PCR is negatively affected by the complex structure of
food and thus its sensitivity decreases depending on food types.
Also, food may include some PCR inhibitors (Perez et al., 2003;
Vaneechoutte & Van Eldere, 1997) which in the end inuence
amplication efciency or primer binding. Especially foods that
have high fat or protein content may cause such decrease in efciency. Thus, culture enrichment becomes a necessity to come up
with this problem. Similarly, the other issue in PCR application is
the DNA purication step. It is performed in PCR assays to x
amplication efciency. Extraction step is crucial and thus should
be done in a careful manner to control all the components and
variables that inuence PCR results. Internal amplication controls
are thus required (Hoorfar et al., 2003) which includes primers that
are specic to a non-target DNA. For instance, a PCR result bringing
out only the 16S rDNA amplicon with the primers specic for 16S
rDNA would represent an effective extraction.
4. Recently developed detection methods
The general requirements of a recent detection method are (i)
increased specicity, (ii) high-throughput results, (iii) increased
reliability, (iv) being applicable in all international laboratories, (v)
having protocols that permits standardization, (vi) rapid technique,
(vii) low cost compared to its alternatives. Considering the demands, immunology-based methods and several types of biosensors are the new developing techniques for detection of

F. Yeni et al. / Food Control 40 (2014) 359e367

foodborne pathogens (Velusamy, Arshak, Korostynska, Oliwa, &


Adley, 2010).
4.1. Sampling and isolation procedures
The sampling protocol does even not exist for recently developed detection methods. The suspensions (containing peptone
water or washing water of a produce) gathered from homogenized
sample or only adjoining the sensor to vegetable/fruit surface is
sufcient (Ercole, Del Gallo, Mosiello, Baccella, & Lepidi, 2003; Li
et al., 2010; Ogunjimi & Choudary, 1999).
4.2. Detection procedures
For immunodetection, which is based on antigen-antibody
binding selection, several types of antibodies can be used; conventional and heavy chain antibodies, as well as polyclonal,
monoclonal or recombinant antibodies. For instance, detection of
L. monocytogenes can be performed via polyclonal antibodies
(Feldsine, Lienau, Forgey, &Calhoon, 1997; Jung, Frank, & Brackett,
2003) and via monoclonal antibodies (Mattingly et al., 1988), but
for Salmonella detection, monoclonal antibodies (Schneid, Ludtke,
Diel, & Aleixo, 2005) have been used. Polyclonal antibodies have
low cost and can be prepared quickly compared to its alternatives
but it has low specicity and abundance (Leonard et al., 2003).
Monoclonal antibodies, on the other hand, have been found to be
more specic and thus used in an extended range of foodborne
pathogens such as L. monocytogenes, Salmonella spp., S. aureus, E.
coli O157, and Shigella. But it has some disadvantages too, requiring
skilled workers, specialized specimen and high cost. Immunological detection methods have been studied on various techniques
such as enzyme immunoassay (EIA) (Borck, Stryhn, Ersboll, &
Pedersen, 2002), enzyme-linked immunosorbent assay (ELISA) (R.
Bennett, 2005), immunochromatography (ICG) strip test (Shim
et al., 2007), immunomagnetic separation (Hudson, Lake, Savill,
Scholes, & McCormick, 2001) etc.
ELISA (enzyme-linked immunosorbent assay) is the most
prominent one among other types of immunological pathogen
detection methods. It integrates the specicity of antibodies and
the sensitivity of simple enzyme assays by using antibodies or
antigens connected to an enzyme (Lazcka, Del Campo, & Munoz,
2007). The enzymes used in ELISA may differ, but the general
ones are alkaline phosphatase, horseradish peroxidase (HRP) and
beta-galactosidase. For instance, detection of L. monocytogenes, E.
coli and C. jejuni is achieved with horseradish peroxidase enzyme
by labeling the antibody for these pathogens that is performed by
sandwich ELISA (Chemburu, Wilkins, & Abdel-Hamid, 2005).
Biosensors, being a rapid method compared to its alternatives
(culture-based methods, molecular methods and immunological
methods), are analytical instruments that works by trapping a
molecule as a reactive surface in close distance to a transducer.
Transducers, which can be in the form of piezoelectric crystals,
electrochemical and optical devices, and acoustic waves, convert
the binding of analyze to the capturing molecule into measurable
signal (Velusamy et al., 2010). The main benets of biosensor
technology are its specicity, sensitivity, reliability, portability, realtime analysis and simplicity of the operation (DSouza, 2001). The
classication of biosensors can be done based on their bio receptor
and transducer types. Enzymes, nucleic acid-based molecules, antibodies, cell/cellular components, biomimetic molecules and bacteriophages can be used as a bio receptor for biosensors. And for
transducer element, optical (Fouriertransform infrared eF-TIR-,
Raman spectroscopy, ber optics, surface plasmon resonance e
SPR-, etc.), electrochemical (amperometrics, potentiometric,

365

conductiometric, etc.) and mass-based (piezoelectric, magnetoelastic, etc.) technologies are studied so far.
Electrochemical methods integrated with magnetic separation
were experienced to detect Salmonella Typhimurium (Che, Yang, Li,
Paul, & Slavik, 1999) and E. coli (Perez, Mascini, Tothill, & Turner,
1998). The methods were completed in less than 2 hours. And the
detection limit was 5  103 cell ml1 for the Salmonella and
105 cell ml1 for the E. coli. Ercole have studied the application of an
polyclonal antibody based biosensor for the detection of E. coli cells
in different six packages of commercial ready to use (RTU) vegetable salads, consisting of rucola, lettuce, carrots and three samples
of mixed-salad (Ercole et al., 2003). The detection limit of the study
was 10 cells/ml and the detection time was 10e20 times smaller
than conventional methods, thus antibody based biosensor detection was designated as a sensitive and rapid technique. In a similar
study, a novel biosensor grounded on electrochemical sandwich
immunoassay for E. coli O157:H7 has been tried on detection in
fresh produce such as lettuce, alfalfa sprouts, and strawberries
(Muhammad-Tahir & Alocilja, 2004). The detection limit of proposed biosensor was estimated to be 81 cfu ml1 and the time for
biosensor detection was as short as 6 min. Similarly, Salmonella
Typhimurium detection has been studied on fresh tomato surfaces
using phage-based magnetoelastic (ME) biosensors which is made
up of a ME resonator platform coated with lamentous E2 phage (Li
et al., 2010). It was concluded that the biosensor was able to detect
5  102 cfu ml1 and higher concentration of Salmonella on tomato
surface in 30 min.
4.3. Advantages & disadvantages of recently developed detection
methods
Immunological methods have the capability of detection of
bacterial cells, spores, viruses and also toxins (especially mycotoxins) (Iqbal et al., 2000). And also they use rapid and more robust
techniques compared to molecular detection methods (Velusamy
et al., 2010).
The sensitivity of biosensor detection methods is similar to
conventional methods (Ercole et al., 2003; Muhammad-Tahir &
Alocilja, 2004). The assay time is decreased to hours or even minutes compared to conventional methods which require days to
detect a foodborne pathogen.
The immunological-based detection is less specic and sensitive
than nucleic acid-based detection.
Biosensors are promising techniques when their rapid and
sensitive detection are taken into consideration but research and
improvement is required to become a reliable and usable alternative (Lazcka et al., 2007). There are too many types of biosensors,
thus there is also a need of standardization for the protocols to be
specic to different pathogen species and food types. Subjects like
being useable in all laboratories, low maintenance, continuous
operation and cost effectiveness should also be needed to be
considered.
5. Conclusions
The key role of rapid and reliable methods have been emphasized by the epidemiological studies of the major outbreaks
occurred in the recent years including the E.coli O104:H4 outbreak
in 2011. These outbreaks entailed emerging of new rapid commercial methods. However, the regulatory agencies and the food
industry have been facing the dilemma of choosing the right
method in screening practices and in emergency situations: using
the rapid methods as screening tools despite the threat of falsenegative results, or using the conventional methods for investigations of foodborne outbreak despite long time requirements.

366

F. Yeni et al. / Food Control 40 (2014) 359e367

At this point, validation of commercial methods is essential and


only a few international institutions perform this task.
Consequently, it is evident that the process of outbreak investigation should be shortened in order to reduce the pathogen
spread and nd the source of contamination by standardized rapid
methods. Extended use of these standardized methods in all
around the world depends on their acceptance by national and
international agencies. Therefore, it is crucial that the process of
validation should be accelerated and the number of international
institutions validating these methods should be increased in order
to have rapid standardized detection methods that can be used
worldwide. Thus, the ultimate goal in the near future should be
developing a rapid and reliable detection method which can be
preferred to time-consuming conventional methods and can be
used as a standard method in all around the world.
Acknowledgment
The research leading to these results has received funding from
the European Union Seventh Framework Programme (FP7/2007e
2013) under grant agreement no: 261752 Plant and Food Biosecurity (PLANTFOODSEC).
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