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Review of Salmonella detection and identification methods: Aspects of rapid

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DOI: 10.1016/j.foodcont.2014.07.011

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 Food Control 47 (2015) 264e276

Contents lists available at ScienceDirect

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

Review

Review of Salmonella detection and identification methods: Aspects of

rapid emergency response and food safety
Kyung-Min Lee a, *, Mick Runyon a, Timothy J. Herrman a, Robert Phillips b, John Hsieh a
a
Office of the Texas State Chemist, Texas AgriLife Research, Texas A&M University System, College Station, TX 77841, United States
b
Food Safety and Inspection Service, U.S. Department of Agriculture, Athens, GA 30605, United States

a r t i c l e i n f o a b s t r a c t

Article history: Salmonella has been recognized as a major and important foodborne pathogen for humans and animals
Received 14 May 2014 over more than a century, causing human foodborne illness as well as high medical and economical cost.
Received in revised form Accordingly, the effort to develop efficient and reliable Salmonella detection methods continues. This
30 June 2014
paper reviews and describes the development and application of commercially available Salmonella
Accepted 3 July 2014
Available online 11 July 2014
detection methods. These are categorized into several groups based on the principle applied: conven-
tional culture methods, immunology-based assays, nucleic acid-based assays, miniaturized biochemical
assays, and biosensors. Conventional culture methods serve as the basis in food testing laboratories
Keywords:
Salmonella detection method
despite rather laborious and time-consuming protocols. Considerable progress in rapid methods using
Food emergency response emerging technologies yield faster answers and higher throughput of samples. This paper also shows and
Food safety analyzes Salmonella test results and summarizes the features and limitations of the studies involving
Foodborne illness Salmonella detection methods developed for emergency response, mainly by food emergency response
Public health laboratories participating during recent fiscal years. The emergency response laboratories utilize Sal-
monella detection methods possessing properties that include simplicity, versatility, high sensitivity,
good specificity, and cost efficiency. Collaboration of the food emergency response laboratories in the
development of these technologies is important essentially to compare for the purpose of continually
improving Salmonella detection methods.
© 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Conventional Salmonella detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Rapid Salmonella detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.1. Immunology-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.1.1. Enzyme-linked immunoabsorbent assay (ELISA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.1.2. Latex agglutination assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.1.3. Immunodiffusion assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.1.4. Immunochromatography (dipstick) assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2. Nucleic acid-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2.1. Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2.2. DNA probe hybridization assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.3. Miniaturized biochemical assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.4. Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.4.1. Enzyme biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.4.2. Nucleic acid biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.4.3. Antibody-based biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.4.4. Micro- and nano-biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

* Corresponding author. Tel.: þ1 979 845 1121; fax: þ1 979 845 1389.
E-mail address: kml@otsc.tamu.edu (K.-M. Lee).

http://dx.doi.org/10.1016/j.foodcont.2014.07.011
0956-7135/© 2014 Elsevier Ltd. All rights reserved.
 K.-M. Lee et al. / Food Control 47 (2015) 264e276 265

4. Salmonella detection methods for food emergency response laboratories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

5. Selection of Salmonella detection methods for rapid emergency response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.1. Matrix effect and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.2. Selectivity and specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.3. Analysis time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.4. Cost efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6. Future aspects of Salmonella detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Uncited reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1. Introduction Hoorfar, 2010; NordVal, 2009). Such standards are critical to

ensure that the results are consistent and comparable among lab-
Salmonella bacteria represent the most common and primary oratories. Several countries have built regulations and guidelines,
cause of food poisoning in many countries for at least over 100 such as Regulation (EC) No 2160/2003, the EU Zoonoses Monitoring
years (Alakomi & Saarela, 2009; Chalker & Blaser, 1988; Coburn, Directive (2003/99/EC), and the Food and Drug Administration
Grassl, & Finlay, 2007). Despite well-established instructions and (FDA) Food Code (EC, 2003a, 2003b; FDA, 2009). These regulations
measures for preventing salmonellosis (Salmonella food poisoning), and guidelines provide or require information on Salmonella and
the incidence and severity of human salmonellosis have signifi- control methods as well as help develop the food safety rules and
cantly increased. Most salmonellosis cases are self-limiting regulatory policies, in order to reduce Salmonella pathogens at any
(approximately 80%), but large outbreaks caused in schools, hos- stage of feed and food production (de Jong & Ekdahl, 2006; Tietjen
pitals, and restaurants are not very common (Guiney et al., 1995). At & Fung, 1995).
present, over 2500 serotypes of Salmonella have been reported Bioterror events, such as anthrax spores in contaminated letters,
(Fierer & Guiney, 2001). Of these, the most common serotypes have greatly altered the view of the public and the scientific com-
associated with human illness are Salmonella enterica serovar munity about bacterial pathogens which could be used as weapons
typhimurium (S. Typhimurium) and S. enterica serovar Enteritidis of biological terrorism, and about the needs and requirements of
(S. Enteritidis) in the United States and European countries. the biotechnology for detection of potential bioterrorism-
Approximately, 1.4 million human Salmonella infections occur employed pathogens (Cannons, Amuso, & Anderson., 2006). Sal-
annually in the United States with assumption of only 2% cases monella spp. is one of such bacterial pathogen which has been
reported to Center for Disease Control and Prevention (CDC), purposely spread mainly using food as a carrier (Ashford et al.,
resulting in about 16,000 hospitalizations with nearly 600 deaths 2003; To€ro€k et al., 1997). For example, S. Typhimurium was used
(Cummings et al., 2010; Mead et al., 1999; Turner, 2010). The esti- to infect residents by contaminating salad bars at the local res-
mated total cost associated with Salmonella incidences may be up taurants in Oregon. The United States capability to respond to a
to several billion dollars annually (Frenzen et al., 1999; WHO, 2005). bioterrorism event has been enhanced by integrated laboratory
An estimated annual cost for a Salmonella control program has networks including the Food Emergency Response Network (FERN)
increased in some countries (WHO, 2005). Much higher incidences and Laboratory Response Network (LRN) of Centers for Disease
related to Salmonella may occur in some developing countries Control and Prevention (CDC) as well as to emerging infectious
where relevant data is not readily available. diseases.
Salmonella is not considered to be fatal to healthy people or as a The purpose of this paper is to review Salmonella detection
bioweapon agent. However, efforts have been made to develop and methods including detection level, sensitivity, specificity, and
improve detection technologies for this organism because Salmo- sample matrices. Included in the discussion are a summary of Sal-
nella may cause devastating foodborne illness. Salmonella detection monella testing results and information from food emergency
methods are desirable to have sensitivity enough to detect one cell response laboratories available in a web-based database portal for
in a defined sample. The analysis time of conventional and rapid recent fiscal years.
methods can vary with cell enrichment steps to reach minimal cell
concentration enough for Salmonella detection. The cell enrichment 2. Conventional Salmonella detection methods
process is typically lengthy in a conventional method whereas the
rapid detection method generally requires at least 104 cells ml1 of Traditional isolation of Salmonella spp. involves a nonselective
Salmonella concentration for detection. pre-enrichment of a defined weight or volume of the sample, fol-
Salmonella surveillance and monitoring should be based on lowed by a selective enrichment step, plating onto selective agars,
reliable and efficient detection methods, which should help and biochemical and serological confirmation of suspect colonies
improve the food safety (Rodríguez-La
zaro et al., 2007). It is (Fig. 1). Different approaches of Salmonella enrichment using the
essential that surveillance and monitoring should cover the entire unique biochemical physical properties of the organisms have been
food chain, preferably starting from investigation of feed and feed standardized by several regulatory agencies, such as International
ingredients for Salmonella contamination (Alakomi & Saarela, Organization for Standardization (ISO), Association of Official
2009; Mead et al., 2010). Standardization, regulation, and interna- Analytical Chemists (AOAC), FDA, and Food Safety and Inspection
tional surveillance networks are also necessary to effectively pre- Service (FSIS) of USDA. The current ISO horizontal method, ISO
vent and control Salmonella pathogens. International Standards for 6579:2002, consists of a pre-enrichment of samples in buffered
the Salmonella tests such as ISO 6579:2002 (Amd 1:2007 Annex D) peptone water (BPW) followed by a selective enrichment in Rap-
and the Nordic Committee on Food Analysis (NMKL) contain in- paporteVassiliadis (soya base) (RVS) and Muller-Kauffmann Tet-
formation about sample storage, sampling, and other critical steps rathionate-Novobiocin (MKTTn) (ISO, 2002). Standard methods
in Salmonella analysis (Feldsine et al., 2003; Lo €fstro
€ m, Hansen, & published for detection of Salmonella by other regulatory agencies
266 K.-M. Lee et al. / Food Control 47 (2015) 264e276
Fig. 1. Scheme of Food Emergency Response Network (FERN)/Food and Drug Admin-
istration (FDA) BAM and Salmonella culture method. RV, RappaporteVassiliadis me-
dium; BS, bismuth sulfite agar; HE, Hektoen enteric agar; XLD, xylose lysine
desoxycholate agar; TT, tetrathionate broth, TSI, Triple sugar iron agar, and LIA, Lysine
iron agar.
are essentially similar to ISO 6579:2002. The conventional cultural
methods for Salmonella recovery basically include the following
essential steps.
Pre-enrichment uses a nutritious nonselective medium to
recover sublethally injured Salmonella cells while inhibiting the
growth of competing flora (Tietjen & Fung, 1995). Most commonly
used media in pre-enrichment step are BPW and lactose broth. The
pre-enrichment process and formulas have been tested to increase
the sensitivity of Salmonella detection. Antimicrobial resistant Sal-
monella spp. could be highly recovered by using appropriate anti-
biotics in pre-enrichment and selective media (Carrique-Mas &
Davies, 2008). The incubated pre-enrichment media are inocu-
lated into selective media containing two or more inhibitory re-
agents such as bile salts, brilliant green, thiosulphate, deoxycholate,
malachite green, novobiocin, tetrathionate, cycloheximide, nitro-
furantoin, and sulphacetamide (Arroyo & Arroyo, 1995; Ha, Pillai, &
Ricke, 1995; Ricke, Pillai, Norto, Maciorowski, & Jones, 1998). The
use of inhibitors in a selective media allows continuous growth of
Salmonella while suppressing the propagation of other bacteria
(Tietjen & Fung, 1995). RappaporteVassiliadis (RV) medium and
tetrathionate (TT) broth has been used as official Salmonella
enrichment media in approved standard methods such as FDA
Bacteriological Analytical Manual (BAM) and FERN Salmonella
methods. Enrichment media has been modified to enhance Sal-
monella growth and thus to increase the sensitivity and selectivity.
The selective enrichment media containing Salmonella at the
level of above 104 cells ml1 are streaked on solid selective media to
isolate presumptive positive Salmonella colonies, simultaneously
inhibiting the growth of other bacteria (Carrique-Mas & Davies,
2008; Ruiz et al., 1996; Tietjen & Fung, 1995). Commonly used
plating media include Salmonella-Shigella agar (SS), brilliant green
agar (BGA), bismuth-sulfite agar (BSA), Hektoen enteric (HE), and
xylose-lysine-deoxycholate agar (XLD). The colonies of different
Salmonella serotypes are differentiated by colors with the coliforms
on these media (Mallinson et al., 2000; Ruiz et al., 1996). For
example, S. Typhi on SS may appear as colorless colonies with black
center. Lactose-fermenting S. Arizonae serotype producing light
pink-red halo around colonies can be also identified on XLD, but
other lactose-fermenting serotypes such as S. Montevideo and S.
Virchow may not be detected on the same agar medium. However,
some serotypes are not distinctive and even missed on those media,
yielding false negatives and increasing cost for additional tests
(Carrique-Mas & Davies, 2008; Gruenewald, Henderson, & Yappow,
1991; Manafi, 2000; Reid, Porter, & Ball, 1993; Ruiz et al., 1996). The
recovery and selectivity of Salmonella may be further enhanced by
using two or more selective media or adding appropriate supple-
ments to plating media such as novobiocin, malachite green, thio-
sulphate, and sulphamethazine (Arroyo & Arroyo, 1995; Carrique-
Mas & Davies, 2008; Kang & Fung, 2000; Tietjen & Fung, 1995).
Presumptive Salmonella colonies isolated on plating media are
typically incubated in triple sugar iron agar (TSI) and lysine iron
agar (LIA) followed by urease test and additional tests for urease-
negative cultures. TSI and LIA media use glucose-fermentation
and lysine decarboxylase reactions for screening Salmonella spp,
respectively. The cultures giving typical reactions for Salmonella are
submitted for biochemical and serological identification tests.
Serotyping identifies the serogroups of the cultures by antigenic
analysis using agglutination reaction subject to antigenic structure.
However, the same Salmonella serotype can vary with antigenicity
due to change and loss of the surface antigens, reducing the
sensitivity of serological methods (Hoorfar, Aherns, & Rådstro € m,
1999; Sørensen, Alban, Nielsen, & Dahl, 2004). If Salmonella iso-
lates cannot be serotyped under certain conditions, PFGE charac-
terization might be useful in place of serological assays.
The conventional microbiological methods serve as the basis for
analysis in many food safety and public health laboratories due to
the ease of use, reliability of results, high sensitivity and specificity,
and lower cost compared to emerging molecular-based technolo-
gies (Gracias & McKillip, 2004; Maciorowski, Herrera, Jones, Pillai,
& Ricke, 2006). However, these procedures need to prepare mul-
tiple subcultures required for several identification steps, taking
more than 5 days for complete isolation and confirmation. In
addition, false positive results may occur due to competitive flora
(e.g. proteus) (Naravaneni & Jamil, 2005; Swaminathan & Feng,
1994). Under circumstances in which high throughput screening
is required for a large number of samples, the laborious and time-
consuming culture-based techniques may not properly address
such a requirement.
The conventional methods have been improved to reduce cost
and labor and to offer faster detection and identification of Sal-
monella. For example, chromogenic and fluorogenic growth media
(e.g. SM-ID agar, Rambach agar, and BBL CHROMagar Salmonella)
used for detection, enumeration, and identification directly on the
isolation plate have shown to be convenient, reliable, and more
specific and selective than conventional media (Alakomi & Saarela,
2009; Maciorowski et al., 2006; Manafi, 2000; Perry & Freydie
re,
2007). The test result using these selective media is typically
available 1 day earlier than conventional methods but is not fast
enough to respond to bioterrorism events, Salmonella outbreak, and
product recall.
3. Rapid Salmonella detection methods
Several rapid methods using molecular cloning and recombi-
nant DNA have been developed, validated, and are available on the
market. These accelerated Salmonella detection procedures enable
quick answers, reduce storage space of conventional supplies and
samples, and increase the throughput of samples (Alakomi &
 K.-M. Lee et al. / Food Control 47 (2015) 264e276 267

Table 1 Table 1 (continued )

A list of Salmonella detection methods commercially available for detection of Sal-
monella spp. Method and Formata Assay Manufacturer

a Tecra® Salmonella Tecra

Method and Format Assay Manufacturer
UNIQUE™
Media Biosensor DIA/PRO UMEDIK
Selective culture BBLTM CHROMagar™ BD Diag. RBD3000 AATI
EF-18 Agar (ISO-GRID Neogen DETEX Moluecular
Method) Circuitry
HGMF/EF-18 QA Life Sci. IMS Dynabeads® anti- Dynal
OSRT (Oxoid Salmonella Oxoid Salmonella
Rapid Test) M1M analyser BioVeris
Oxoid SRT Oxoid PATHATRIX Matrix
Rambach agar Technogram MicroScience
RAPID' Salmonella Bio-Rad Lab. Salmonella Screen VICAM
S.P.R.I.N.T. Salmonella Oxoid Agglutination TUBEX IDL Biotech
Salmonella Contamination Blot CheckPoint Colony Lift Kirkegaard &
Sciences LLC Immunoassay Kit Perry
Salmonella Precis Oxoid Dot-ELISA TyphiDot™ Moody
Salmonella Rapid Test Oxoid ECL PATHIGEN BioVeris
SESAME Salmonella Biokar Diag. Immunodiffusion Salmonella 1-2 Test BioControl
Test Lateral flow Multi-Test Dip-S-Ticks PANBIO INDX
Simple Mehtod for AES Chemunex Tube agglutination Sanofi qualitative Bio-Rad
Salmonella (SMS) agglutination
Suncoli Test Paper Sun Chem. Nucleic acid-based
Immunology-based PCR ABI Prism 7500 Applied
ELISA Assurance GDS™ for BioControl Biosystems
Salmonella ADIAFOOD Salmonella AES Chemunex
BacTrace KPL Assurance GDS Biocontrol
Salmonella ELISA Test Bioline Salmonella Systems
SELECTA/OPTIMA BAX Salmonella PCR DuPont
CHEKIT Bommeli Diag. Qualicon
Colortrix Matrix AmpliSensor Biotronics
\Microscience Foodproof Salmonella Merck KGaA
EIAFoss FOSS detection kit
Equate Binax foodproof® Salmonella BIOTECON
FlockChek IDEXX Detection Kit Diagnostics
Immuno-magnetic- Golden GeneDisc Salmonella Pall
separation spp. GeneSystems
Locate Rhone-Poulenc GeneSTAR Gull
MASTAZYME MAST Laboratories
MicroELISA Dynatech Lab. Genevision Warnex
Rapidyme Salmonella BIO ART SA/NV Gene-Trak Neogen
Ridascreen Salmonella R-Biopharm HQS Salmonella ADNucleis
Salmonella GEM iQ-Check™ PCR BioRad
Salmonella Dioline/Mast Diag. Laboratories
Salmonella-TEK Organon Teknika LightCycler Roche
Salmotype Labor Diag Leipzig Diagnostics
TAG 24 Salmonella Biocontrol Luminex Luminex
Tecra® Salmonella Tecra Corporation
VIA™ MicroSEQ Salmonella Applied
TRANSIA Salmonella Diffchamb AB spp Biosystems
VIDAS bioMe
rieux Probelia Sanofi
LA ANI Salmonella ANI Biotech Diagnostics
Bactigen Wampole Lab. Pasteur
DuPont Lateral Flow DuPont Qualicon SmartCycler Cepheid
System TagMan PE-Applied
Microscreen Microgen Biosystems
RapidTest Unipath DNA hybridization DNAH GeneTrak
RapidChek SELECT SDIX GeneQuence™ Neogen
Salmonella Corporation
Salmonella Seiken Denka Seiken Gene-Trak® Neogen
Salmonella Verify VICAM Corporation
Serobact REMEL RABIT DonWhite Sci
Singlepath Salmonella EMD Miniaturized tests
Salmonella Latex test Oxoid Biochemical API 20E bioMe
rieux sa
Salmonella Screen/ Vicam Enterobacteriaceae Set II BBL
Salmonella Verify Enterotube II Roche Diag.
Slidex bioMe
rieux EPS (Enteric Pathogens Vitek
Spectate May & Baker Screen Card)
VIP® Salmonella BioControl GNI (Gram Negative Vitek
Wellcolex Wellcolex Identification Card)
Immunochromatography Salmonella 1-2 SE BioControl MICRO-ID Organon Teknika
Clearview Unipath Microscan Baxter Diag.
PATH-STIK Celsis Mnitek I1 BBL
Reveal® for Salmonella Neogen Quad Enteric Panel Micro-Media
Singlepath Merck Quantum I1 Abbott Lab.
SMART-II New Horizon (continued on next page)
268 K.-M. Lee et al. / Food Control 47 (2015) 264e276
Table 1 (continued )
Method and Formata Assay Manufacturer
Sensititre Sensititre
Tri Panel DifcoPasco Lab.
Vitek 2 bioMe
rieux
a Salmonella spp. is bound to the appropriate antibody linked to a
ELISA, enzyme-linked immunoabsorbent assay; LA, latex agglutination; IMS,
immunomagnetic separation; ECL, electrochemiluminescence, PCR, Polymerase
chain reaction.
Saarela, 2009; Blivet, Soumet, Ermel, & Colin, 1998; Tietjen & Fung,
1995). The rapid method may be defined as one which allows the
detection of Salmonella spp. in samples and delivers reliable results
within a few hours to a day (Ferretti, Mannazzu, Cocolin, Comi, &
Clementi, 2001; Tietjen & Fung, 1995).
Commercially available rapid methods for Salmonella detection
can be divided into several categories including new selective
media, modified or adapted conventional procedures,
immunology-based assays, and nucleic acid-based assays (Alakomi
& Saarela, 2009; Blivet et al., 1998; Eijkelkamp, Aarts, & van der
Fels-Klerx, 2009; Iqbal et al., 2000; Tietjen & Fung, 1995)
(Table 1). Of these methods, ELISA and PCR procedures show
comparable specificity and sensitivity to conventional methods.
ELISA assays are able to detect Salmonella concentration at the level
of 104e105 ml1 while PCR-based assays provide the level of
sensitivity of 104 ml1 after enrichment. The sensitivity and spec-
ificity of these methods largely depend on the background micro-
flora, sample matrix, presence of non-culturable cells, and
inhibitory substances (e.g. fats, proteins, polysaccharides, heavy
metals, antibiotics, and organic compounds) (Alakomi & Saarela,
2009; Mozola, 2006; Naravaneni & Jamil, 2005). It has been re-
ported that the sensitivity and detection limits of the methods were
improved by additional sample preparation and purification tech-
niques such as modification of pre-enrichment and enrichment
media, dilution, centrifugation, filtration, flow injection, chroma-
tography, organic solvent extract, and fluorescence hybridization
(Kutter, Hartmann, & Schmid, 2006; Mozola, 2006; Polaczyk et al.,
2008; Wolffs, Glencross, Thibaudeau, & Griffiths, 2006).
3.1. Immunology-based assays
Immunology-based assays which employ specific mono- or
polyclonal antibodies binding with somatic or flagella antigens
have been broadly used for detection of Salmonella spp. in a variety
of sample matrices (Betts, 2002; Blivet et al., 1998; Iqbal et al.,
2000; Maciorowski et al., 2006). The immunology-based assays
include enzyme-linked immunoabsorbent assay (ELISA), latex
agglutination tests, immunodiffusion, and immunochromatog-
raphy (dipstick). As routine analysis techniques, these assays
remain a valuable option due to the ability to detect viable non-
culturable Salmonella cells (Maciorowski et al., 2006). The previ-
ous studies showed that the immunology-based assays are com-
parable to and more rapid and specific than cultural methods,
feasible to isolate Salmonella spp. in conjunction with immuno-
magnetic separation (IMS) techniques to handle large samples, and
readily automated to reduce time and labor (Keith, 1997; Tietjen &
Fung, 1995; Uyttendaele, Vanwildemeersch, & Debevere, 2003).
However, these methods have some potential shortcomings for
detection of Salmonella including a longer enrichment time to
obtain the appropriate number of cells, cross-reactions with closely
related antigens, antigen variation, limits in sensitivity for some
sample matrices and stressed cells, and high cost for automation
(Blivet et al., 1998; Bolton, Fritz, & Poynton, 2000; Dill, Stanker, &
Young, 1999; Uyttendaele et al., 2003).
3.1.1. Enzyme-linked immunoabsorbent assay (ELISA)
Of immunology-based assays, ELISA is the most commonly used
assay for the detection of antigens or products of Salmonella spp.
The different ELISA systems have been developed and commer-
cially available in kit form. In the ELISA assay, an antigen specific to
solid matrix. After forming the antigeneantibody complex, the
concentration of the antigen and the presence of Salmonella can be
measured through the change in color caused by the enzymatic
cleavage of a chromogenic substrate (Blivet et al., 1998; Tietjen &
Fung, 1995). Alternatively, the presence of antibodies in samples
infected with Salmonella spp. can be detected using antigens
coupled to the solid phase of ELISA (Wiuff et al., 2000). ELISAs have
also been used to detect antibodies for development of vaccines
against Salmonella infections (Meenakshi et al., 1999). Several
commercial validated ELISA systems designed for rapid detection of
Salmonella in raw or processed products are available: Assurance
GDS™ for Salmonella (BioControl Systems, Inc., Bellevue, WA),
TECRA Salmonella (Tecra International Pty Ltd, French Forest, New
South Wales, Australia), Salmonella ELISA Test SELECTA/OPTIMA
(Bioline APS, Denmark), and Vitek Immuno Diagnostic Assay Sys-
tem (VIDAS) (BioMerieus, Hazelwood, MO) (Table 1). The aggluti-
nation technique employs latex particles coated with antibodies
which react with antigens on the surface of Salmonella cells to form
visible aggregates for identification of Salmonella positive samples
(Thorns, McLaren, & Sojka, 1994; Tietjen & Fung, 1995). The assays
are specific, uncomplicated, and reliable so that generally, they
have been used as a confirmatory analysis technique, rather
screening test for Salmonella organisms (Eijkelkamp et al., 2009;
Love & Sobsey, 2007). There are several commercial kits available
on the market (Table 1).
3.1.2. Latex agglutination assay
The agglutination technique employs latex particles coated with
antibodies which react with antigens on the surface of Salmonella
cells to form visible aggregates for identification of Salmonella
positive samples (Thorns et al., 1994; Tietjen & Fung, 1995). The
assays are specific, uncomplicated, and reliable so that generally,
they have been used as a confirmatory analysis technique, rather
screening test for Salmonella organisms (Eijkelkamp et al., 2009;
Love & Sobsey, 2007). There are several commercial kits available
on the market, including Spectate (May & Baker Diagnostics Ltd.,
Glasgow, Scotland, UK), Wellcolex color Salmonella (Wellcolex,
Merseyside, UK), Salmonella Latex test (Oxoid, Basingstoke, UK),
Bactigen (Wampole Laboratories, Cranbury, NJ), and Slidex (Bio-
Me
rieux, Marcy L'Etoile, France).
3.1.3. Immunodiffusion assays
The Salmonella 1-2 Test system (BioControl Systems, Inc.,
Bothell, WA) for detection of motile Salmonella is a commercial kit
that operates on the basis of immunodiffusion reaction (D'Aoust &
Sewell, 1988; Flowers & Klatt, 1989; Nath, Neidert, & Randall, 1989;
Tietjen & Fung, 1995). Before inoculation into the system unit
which consists of two connected chambers, the sample is pre-
enriched for 24 h. The enriched sample is inoculated to a tetrathi-
onate brilliant green broth in the inoculation chamber. Salmonella
then moves out of the inoculation chamber into the mobility
chamber in which antibody has been added onto a distal surface of
a semisolid medium. Salmonella in the mobility chamber is
immobilized by forming an antigeneantibody complex. After in-
cubation for 14 h, the readable three-dimensional immunodiffusion
band is produced. Modifications in an enrichment step before
inoculation and an increase of incubation time improved the
effectiveness of detection of Salmonella spp. (Nath et al., 1989). The
 K.-M. Lee et al. / Food Control 47 (2015) 264e276
modified and original assays have shown to be equivalent with the
reference culture method.
3.1.4. Immunochromatography (dipstick) assays
The commercial Salmonella test kits, based on the dipstick
format, include the Tecra® Salmonella Unique™ test (Tecra Inter-
national Pty Ltd, French Forest, New South Wales, Australia) and the
PATH-STIK (Celsis, Inc., Edison, NJ). The Tecra Salmonella Unique™
test which consists of the immunoenrichment and detection steps
is a screening procedure for Salmonella detection. The PATH-STIK
Salmonella test is an immunological dipstick assay for one-step
detection of Salmonella. The dipstick contains an assay control
and all reagents used for Salmonella detection. Prior to the dipstick
assay, a sample is pre-enriched and selectively enriched in media.
The enriched sample is applied to the test unit and Salmonella in the
sample is captured onto the dipstick. Unlike the Tecra® Salmonella
Unique™ test, the PATH-STIK doesn't include a washing step,
requiring only 30 min for analysis (van Beurden, 1992; Brinkman,
van Beurden, Mackintosh, & Beumer, 1995).
3.2. Nucleic acid-based assays
The nucleic acid-based assays are Salmonella detection tests that
utilize a specific nucleic acid target sequence within the organism.
The assays have been most intensively explored and developed for
the past decade among Salmonella detection methods because they
offer some advantages of sensitivity, specificity, and inclusivity over
other methods, rapidly identifying Salmonella without obtaining
pure cultures (Glynn et al., 2006; Mozola, 2006). Two major tech-
niques of the assays are direct hybridization (DNA probe) and
amplification (PCR) methods. The great progress of the assays al-
lows the detection of very low numbers of organisms in the sample
and high throughput of a large number of samples for routine
analysis (Cohen et al., 1993; Mozola, 2006; Rasmussen, Rasmussen,
Larsen, Hoff-Jorgensen, & Cano, 1994).
3.2.1. Polymerase chain reaction (PCR)
The PCR assay is the preferred non-cultural technique for in vitro
primer-meditated enzymatic amplification of specific segments of
DNA for the detection of Salmonella pathogens (Cocolin, Manzano,
Cantoni, & Comi, 1998; McKillip & Drake, 2004; Wolcott, 1991). The
PCR assay can exponentially amplify a single specific sequence to
one million-fold of DNA fragment within 2 or 3 h using a ther-
mostable DNA polymerase. The amplified products can be detected
by several means (e.g. gel-based systems and real-time PCR)
(Eijkelkamp et al., 2009; Mozola, 2006). The development and
advancement of the technique for amplifying specific segment of
DNA improve the specificity and sensitivity enough for detecting
even one molecule of target DNA in a defined sample. Due to the
capability to detect such a low concentration of Salmonella,
enrichment times are considerably shorter to reach Salmonella
concentration needed for reliable detection by the PCR compared to
other assays. The equivalency was reported between the PCR assay
and the standard culture methods in detecting Salmonella in food
products (Aabo, Andersen, & Olsen, 1995; Cano, Rasmussen,
Sanchez Fraga, & Palomares, 1993; Eyigor, Carli, & Unal, 2002;
Stone, Oberst, Hays, McVey, & Chengappa, 1994). The PCR assays
have been developed, validated, and standardized according to the
general guidelines and requirements by ISO (IOS 20838:2006, ISO/
DIS 22119, ISO 16140:2003, ISO 6579:2002, ISO 22174).
Commercial kits based on the PCR technology have been
developed, and successfully used for routine Salmonella screening
in the industry and emergency response laboratories. The PCR-
based commercial kits and systems with different specificity and
sensitivity include ABI Prism 7500 (Applied Biosystems,
269
Warrington, UK), Probelia (Sanofi -Diagnostics Pasteur, Marnes-la-
Coquette, France), BAX system (DuPont Qualicon, Wilmington, DE),
TagMan (PE-Applied Biosystems, Foster City, CA), Gene-Trak (Neo-
gen Corporation, Lansing, MI), iQ-Check™ PCR (BioRad Labora-
tories, Hercules, CA), LightCycler (Roche Diagnostics, Manheim,
Germany), and SmartCycler (Cepheid Inc., Sunnyville, CA). The BAX
system is the first commercially available PCR method using a
single tablet combining all reagents (primers, enzyme, and de-
oxyribonucleotides) needed for PCR to rapidly amplify and detect
Salmonella spp. (Bennett,., Greenwood, Tennant, Banks, & Betts,
1998; Hoorfar et al., 1999; Maciorowski et al., 2006).
The real-time PCR (RT-PCR) system detects accumulated PCR
product by monitoring the increase fluorescence signal using the
integrated real-time thermocycler and fluorescence detector
within a system. This helped overcome the problem of false positive
results posed by amplicon contamination (Maurer, 2011). The false
positive results can be further excluded by an internal standard
used for ensuring the absence of inhibitors. The RT-PCR system
employing automated DNA extraction, amplification, and detection
has improved the ability of industry and emergency response lab-
oratories in identifying Salmonella from a variety of sample
matrices. The RT-PCR methods available on the market can be
classified into several categories based on the principle and
approach of the methods. Several fluorescent techniques have been
developed and incorporated into the RT-PCR systems for simplicity
and cost saving in detecting the target Salmonella sequences.
3.2.2. DNA probe hybridization assay
A DNA probe hybridization assay uses a DNA probe with a
sequence complementary to the target sequence of a DNA or RNA
molecule in the target organism (de Boer & Beumer, 1999; Fung,
2002; Mozola, 2006). In this assay, the target cells are first lysed
and the produced nucleic acids are purified prior to hybridization
with a DNA probe. The unbound probe to a target sequence is
eliminated by washing. The stable hybrid with the labeled DNA can
be detected using different detection techniques, such as radio-
isotopes and enzymatic reactions. The presumptive Salmonella re-
sults are typically confirmed by the culture methods.
The DNA probe hybridization assays have some advantages in
specificity and sensitivity of identifying Salmonella in samples
compared to other methods. The assays are effective to rapidly screen
the large numbers of samples and highly specific to detect the target
cells without cross-reactions with the other organism (Agron et al.,
2001; de Boer & Beumer, 1999; Dunbar & Jacobson, 2007; Mozola,
2006). This may allow more rapid distribution of Salmonella-free
products and more frequent monitoring at important investigation
sites. However, the required sensitivity may be achieved only in the
presence of sufficient concentration of target organisms after pre-
enrichment (Jones, Law, & Bej, 1993). The amplification of target
nucleic acid sequence and DNA probe might be an alternative way to
increase the sensitivity of the assays (Tietjen & Fung, 1995).
The Gene-Trak® Salmonella Assay (Neogen Corporation, Lansing,
MI) is the first introduced commercial assay. The second generation
of this assay is based on a hybridization format of two probes, a
polyadenylic acid (poly dA) tail on the capture probe and a fluo-
rescein labeled detector probe, to the Salmonella ribosomal RNA
(rRNA) and enzyme-mediated colorimetric detection. The Gene-
Trak® Salmonella Assay has been modified to the direct-labeled
format which replaces a fluorescein labeled detector probe with
HRP. This assay is equivalent to the original dipstick format assay
and greatly simplifies the procedure by eliminating the enzyme
incubation and washing steps and by lowering a hybridization
temperature (Eijkelkamp et al., 2009; Mozola, 2006). The microwell
format assay was also introduced and available on the market un-
der the name GeneQuence™ Salmonella.
270 K.-M. Lee et al. / Food Control 47 (2015) 264e276
The different colorimetric DNA hybridization assays available in
the market have less than 48 h of a test time frame. Since the assays
require higher detection limits of enrichment culture for positive
signal, they need a longer enrichment time to increase the con-
centration of Salmonella in the final culture. However, the total test
time with certain samples can be shortened to a significant extent.
GeneQuence® Salmonella showed a sensitivity of 97.1% using a new
2-stage procedures including 24e27 h pre-enrichment and selec-
tive enrichment followed by a 2 h testing time in selected foods
(Alles, Peng, Wendorf, & Mozola, 2007). The assays based on such
rRNA hybridization approach have certain benefits in designing a
probe because rRNA sequences are highly conserved, stable, and
abundant in a bacteria cell, providing various targets and increasing
the assay sensitivity (Mozola, Peng, & Wendorf, 2007).
3.3. Miniaturized biochemical assay
Miniaturized biochemical assay employs the reduced volumes
of reagents, media, and vessels for rapid characterization of Sal-
monella to replace conventional biochemical tests and to provide
more results in the same span of time (Fung, 2002; Ge & Meng,
2009; Tietjen & Fung, 1995). Many microbiologists have devel-
oped and used diverse miniaturized biochemical systems to rapidly
confirm isolated organisms from a larger number of samples. Most
miniaturized biochemical systems consist of disposable sterilized
microtiter plates, a multiple inoculation device, and 10e20 media
or substrates specially designed to target organisms. Salmonella
spp. are identified based on the assimilation and utilization of
special substrate and automated colony and cell density measure-
ment. The test procedures include placing pure cultures into wells
of a microtiter plate each of which represents independent inocu-
lation in the conventional method, holding up to 96 different cul-
tures. After 16e24 h incubation at a desirable temperature, the
presence or absence of Salmonella on solid media or liquid media
can be determined by reading color changes caused by the reaction
of specific compositions and metabolites with reagents with the aid
of a manual identification code or an automatic reader interfaced
with a computer.
Diverse miniaturized kits for rapid biochemical characteriza-
tion of Salmonella are commercially available, including API 20E
(bioMe
rieux sa, Marcy-I'Etoile, France), Enterotube II (BD Di-
agnostics, Sparks, MD), Enterobacteriaceae Set II (previously
known as Mnitek II, BBL Microbiology Systems, Cockeysville, MD),
MICRO-ID (Remel, Lenexa, KS), EPS (Enteric Pathogens Screen
Card) (Vitek Systems, Hazelwood, MO), GNI (Gram Negative
Identification Card) (Vitek Systems, Hazelwood, MO), Microscan
(Baxter Diagnostics, West Sacramento, CA), Quad Enteric Panel
(Micro-Media Systems, San Jose, CA), Quantum II (Abbott Labo-
ratories, Diagnostic Division, Abbott Park, IL), Sensititre (Sensititre,
Salem, NH), Tri Panel (DifcoPasco Laboratories, Wheat Ridge, CA),
Vitek 2 (bioMe
rieux sa, Marcy-I'Etoile, France), and Walk Away
(Dade MicroScan, West Sacramento, CA). The majority of these
commercial kits were first used for comparative analyses of clin-
ical samples and later applied for food microbiological samples
(Fung, 2002; Stager & Davis, 1992). These systems use similar
approaches and procedures ideal for analyzing large number of
samples and isolates. The sensitivity and specificity of these sys-
tems, particularly the latest ones, easily exceed 95% with the
higher sample throughput, but the analysis cost may significantly
increase if a sophisticated automated identification system is used.
Most miniaturized biochemical assay kits were initially designed
for identification of gram-negative bacteria including Salmonella,
Shigella, and Enterobacteriaceae, and later expanded to gram
positive bacteria, anaerobes, and even yeasts and molds (Gracias &
McKillip, 2004).
The previous studies reported that miniaturized biochemical
systems are accurate, efficient, convenient, labor-saving, space-
saving, and more economical compared to the conventional culture
methods (Cox, Fung, Bailey, Hartman, & Vasavada, 1987; Feng,
1997; Holmes, 1989; Kalamaki, Prioce, & Fung, 1997; Stager &
Davis, 1992). Except for the MICRO-ID and APE 20E system
requiring 4 h of incubation, the results are typically obtained after
24 h of incubation at an appropriate temperature in other systems.
These miniaturized biochemical systems and kits have significantly
contributed to improve accuracy, efficiency, and capacity in
detecting Salmonella spp. It is anticipated that in the future they
will continue to play an important role in the food, industrial, or
environmental microbiology areas.
3.4. Biosensors
Biosensor technology encompasses an applied microbiology
approach to bacterial detection (Alonso-Lomillo, Domínguez-
Renedo, & Arcos-Martínez, 2010; Baeumner, 2003; Fung, 2002;
Lazcka, Campo, & Mun  oz, 2007) that defines a molecule or a
group of molecules by incorporating materials immobilized on the
surface of a physicochemical transducer or transducing micro-
system. A recognition signal is generated when a specific analyte
binds to the biological recognition element. The signal can be
changes in mass, oxygen consumption, potential difference,
refractive index, pH, current, and other parameters (Baeumner,
2003; Eijkelkamp et al., 2009). It is anticipated that a biosensor
technique may replace existing immunology- and nucleic acid-
based assays (Alocilja & Radke, 2003; Lazcka et al., 2007; Van
Dorst et al., 2010).
Biological recognition elements used in biosensor application
include enzymes, antibodies, nucleic acids, whole cells, tissue/
whole organisms, and biomimetic material. The signal recognition
and transduction in the biosensor is achieved by different types of
transducers: electrochemical, optical, thermometric, micro-
mechanical, and other miscellaneous transducers according to
which the biosensors have been often classified by several authors
(Goepel & Heiduschka, 1995; Ivnitski, Abdel-Hamid, Atanasov,
Wilkins, & Stricker, 2000; Leonard et al., 2003).
3.4.1. Enzyme biosensor
An enzyme biosensor is an analytical device that integrates an
enzyme with a transducer to produce a signal resulting from
different physicochemical changes caused by enzyme-catalyzed
reactions. The signal is converted into measurable responses such
as current, temperature change, and light absorption to determine
target analyte concentration (Baeumner, 2003). Enzyme biosensors
are highly selective, rapid, and easy to use while they are expensive
and sometimes lose their activities when immobilized on a trans-
ducer. Enzymes used in this type of biosensor include glucose ox-
idase, galactosidase, glucoamlyase, lactate oxidase, and other
enzymes (Fung, 2002).
3.4.2. Nucleic acid biosensor
A nucleic acid biosensor which consists of a nucleic acid as a
biological recognition element and a signal transducer is based on
the hybridization of complementary strands of DNA or RNA mole-
cules for detection of organisms (Baeumner, 2003; Eijkelkamp
et al., 2009; Lazcka et al., 2007). In addition to detection of bacte-
ria and other pathogens, nucleic acid biosensors have been applied
to food and environmental samples for detection of toxic com-
pounds and biotechnology products using different signal trans-
duction systems such as quartz crystal microbalances, surface
plasmon resonance, surface acoustic wave sensor, and evanescent
wave biosensors (Deisingh & Thompson, 2004; Feriotto, Borgatti,
 K.-M. Lee et al. / Food Control 47 (2015) 264e276
Mischiati, Bianchi, & Gambari, 2002; Mascini, Palchetti, &
Marrazza, 2001).
3.4.3. Antibody-based biosensors
Antibody-based biosensors are analytical devices which use an
antibody as a biological recognition element attached to a signal
transducer to quantify the target analyte concentration (Bilitewski,
2000; Rogers, 2000). Antibodies are the most commonly used
biological recognition element due to their high specificity and
affinity to antigen, diversity, and availability on the market.
Antibody-based biosensors can be divided into three main assay
formats including an immobilized antibody, immobilized antigen,
and non-immobilized antibody or antigen whose characteristics
are largely determined by the type of signal transduction mecha-
nisms (Rogers, 2000).
3.4.4. Micro- and nano-biosensors
The field of micro- and nano-biosensors has been drawing much
attention from microbiologist due to their great potential to solve
many current problems including real time detection of a variety of
pathogens, and believed as one of the future directions to detect
Salmonella spp. (Chen et al., 2005; Mozola, 2006; Scaria et al.,
2008). However, actual application to Salmonella detection is
rather limited so far because of the instrumental complexity and
immature stage of the technology development (Alakomi &
Saarela, 2009). However, the techniques are rather restricted in
application to pathogen detection because of their incompatibility
with some biological samples. Despite some obstacles and chal-
lenges in the research and application of the micro- and nano-
biosensors, rapid development and integration of associated core
technologies seems to provide no doubt that in the future they
would be one of important technologies in microbiology and have a
great value in the market.
4. Salmonella detection methods for food emergency
response laboratories
Salmonella detection is critical for emergency responders who
require robust and preferably transportable laboratory-based
detection systems. The methods for Salmonella detection typically
recommended by the FDA and the FSIS of the USDA are based on
cultural, serological, and biochemical properties using selective
media. However, rapid methods for Salmonella detection have
become increasingly important for effectively monitoring food
products and are considered desirable as a future approach and
national strategy. Rapid test kits recommended by the US Food
Emergency Response Network for Salmonella include the VIDAS
Salmonella (SLM) and immuno-concentration Salmonella (ICS)
methods, the Tecra Unique Salmonella test, and the polymerase
chain reaction (PCR)-based BAX™ system, which were chosen with
the expectation of providing results within 24 h. These test kits are
approved for screening purposes by the AOAC and have been
widely used by food testing laboratories.
During the past four fiscal years, food emergency response
laboratories have performed single lab and/or multi-lab validations
involving rapid Salmonella detection platforms. Several studies
have focused on the recovery of Salmonella spp. from a variety of
sample matrices spiked with Salmonella strains. According to the
results of these studies, detection levels varied with sample matrix
and spiking concentration. For example, when spiked at the same
low concentration Salmonella was easily identified in cat food and
vegetable burgers, while its detection in other matrices such as
baby oatmeal, peanut butter, liquid eggs, and cantaloupe was more
difficult. Several food matrices, including milk, strawberries, and
creamed corn, displayed an inverse relationship between
271
detectability and concentration. Furthermore, wild-type Salmonella
enteriditis showed much lower recoveries from liquid egg products
relative to the American Type Culture Collection (ATCC) strains.
In comparison studies of recovery of Salmonella from culture
media, Yersinia pestis enrichment (YPE) broth and buffered peptone
water (BPW) were as effective as lactose broth, a standard enrich-
ment medium, in some sample matrices. Overall, xylose lysine
deoxycholate (XLD) agar exhibited a higher recovery of Salmonella
spp. than Hektoen enteric (HE) and bismuth sulfite (BS) agars,
which may be explained by the selectivity of the media and the
physiological properties of the strains used in the studies. Sample
preparation techniques also appeared to affect the recovery of the
spiked samples. For example, results obtained with the VIDAS® SLM
and BAX® Q7 Salmonella PCR assays indicated that washing with
soaking and soaking alone were more effective for extraction of
Salmonella than surface washing only.
Comparative and collaborative studies of the rapid methods for
screening of Salmonella have been performed by many food
emergency response laboratories by using a variety of spiked
sample matrices. However, as seen in Table 2, only limited numbers
of methods and platforms were extensively evaluated and
compared within integrated laboratory networks during this spe-
cific period. In the validation studies, a suspension of Salmonella
cells at the appropriate turbidity was serially diluted for spiking
into samples prior to assessing the test kits. Enrichment using BPW
or YPE broth for the PCR-based methods offered a rapid alternative
to the standard culture method for identifying Salmonella spp. Of
the PCR methods tested, the BAX™ system showed comparable
sensitivity and specificity to conventional culture methods in most
sample matrices spiked at low and high concentrations of Salmo-
nella cells, with the exception of some false results due to cross-
contamination of PCR products. No differences in identifying Sal-
monella spp. were found between the standard BAX™ system and
the BAX™ Q7 system.
Overall, there was little or no difference between the rapid
methods, which displayed equally high accuracies in identification
of the target organisms regardless of the matrix. Comparable re-
sults were found between BAX and SmartCycler PCR platforms in
single laboratory testing. Some laboratories reported that the per-
formance and accuracy of the SmartCycler PCR system was some-
what dependent on the characteristics of the sample matrix. It was
also reported that the sensitivity of the systems was further
improved by increasing enrichment time. Taken together, these
observations seem to imply that the difference between these
methods may be mainly due to the matrix properties and the
possible presence of PCR inhibitors, rather than the inherent
effectiveness of the assay systems.
Several laboratories have compared the BAX™ Q7 PCR and
VIDAS Salmonella (SLM) assays. The results showed no significant
difference between the two assays in detecting Salmonella spp. in
most food matrices. Indeed, most failures of the two assays in
Salmonella detection appeared to be related to the matrix effect.
Several laboratories have indicated that the VIDAS assay might be
more reliable than the BAX system based on the study results and
on the discussions moderated through a web-based database por-
tal. However, the VIDAS assay was insufficiently sensitive for
certain matrices (e.g., bean sprouts), and requires a secondary
enrichment procedure to reduce the effects of background flora.
Furthermore, there is only limited information available on how the
BAX™ and VIDAS systems interact with matrices.
Several immunomagnetic separation (IMS) systems such as the
Dynal BeadRetriever, Pathatrix, and BioVeris M1M Platform were
validated and assessed for their efficiency in recovering and
isolating different strains of Salmonella from sample matrices
(Table 2). Typically, after an overnight incubation, a sample aliquot
272 K.-M. Lee et al. / Food Control 47 (2015) 264e276
Table 2
Salmonella detection methods for screening of Salmonella and sample matrices used
for the studies in food emergency response laboratories during the past fiscal years.
Methoda Sample matrix
PCR assays
ABI 7500 Fast Cheese, cookie, cookie, ground beef, ice
cream, liquid egg, peanut butter, potato
salad, cracker, snack bar
BAX® PCR system Baby carrot, beef stick, black pepper,
cilantro, cilantro flakes-dried, cooked
ham, cooked ham, deli ham, ground
beef, ground beef, hamburger, hot
pepper, hot dog, infant formula,
jalapeno pepper, liquid egg, milk,
orange juice, peanut butter, potato
salad, powdered formula, precut ready-
to-eat (RTE) lettuce, serrano pepper,
spinach, sponge sicle, tomato, vegetable
juice, whole head romaine lettuce
LightCycler Alfalfa sprout, baby oatmeal, broccoli,
cantaloupe, cat food, chicken, creamed
corn, frozen spinach, ground beef, hot
dog, jalapeno pepper, lettuce, liquid
egg, milk, orange juice, peanut butter,
strawberry, tomato, vegetable burger,
watermelon
SmartCycler Baby carrot, cheese, chicken breast deli
meat, cookie, deli ham, ground beef, ice
cream, liquid egg, milk, orange juice,
peanut butter, potato salad, raisin, raw
pork sausage, cracker, snack bar,
spinach, vegetable juice
Luminex Alfalfa sprout, chocolate, bean sprout
ELISA assay
VIDAS Cilantro, cilantro flakes-dried, jalapeno
pepper, precut ready-to-eat (RTE)
lettuce, serrano pepper, spinach, whole
head romaine lettuce
IMS assays
Dynal BeadRetriever Chicken breast deli meat, cooked ham,
ground beef, hamburger, liquid egg,
milk, orange juice, peanut butter, potato
salad, raisin, raw pork sausage, spinach
M1M analyzer Alfalfa sprout, baby oatmeal, broccoli,
cantaloupe, cat food, chicken, creamed
corn, frozen spinach, ground beef, hot
dog, jalapeno pepper, lettuce, liquid
egg, milk, orange juice, peanut butter,
strawberry, tomato, vegetable burger,
watermelon
PATHATRIX Hot pepper, peanut butter, tomato
Miniaturized biochemical assays
API 20E Chicken breast deli meat, raisin, raw
pork sausage
Enterotubes Liquid egg, milk, orange juice, peanut
butter, spinach
Vitek® Black pepper, ground beef, peanut
butter, potato salad, powdered formula
Media
CHROMagar Ground beef, liquid egg, potato salad
a sampling and sample preparation have not been optimized for the
PCR, Polymerase chain reaction; ELISA, enzyme-linked immunoabsorbent assay;
IMS, immunomagnetic separation.
was subjected to immunomagnetic separation and the two bacte-
rial fractions (immuno-bound and unbound) were plated onto
agarified medium and/or processed using the Roche LightCycler 2.0
RT-PCR, BAX System Q7 PCR, or Applied Biosystems 7500. Irre-
spective of manufacturer and concentration of Salmonella, the IMS
systems were generally effective for improving the recovery from
sample matrices and allowing for shorter turnaround times.
However, the recovery from some food matrices (e.g., bean sprouts,
cooked ham, ground beef, and peanut butter) was identical with or
without the use of an IMS system at all spiking levels, indicating
that the system efficiency is highly dependent on sample matrix. In
addition, the IMS systems appeared to be not fully functional when
the complexity of the sample matrices increased. Compared to
manual methods, the automated IMS systems further increased the
speed and productivity, and minimized cross-contamination by
significantly reducing sample transfer time and pipetting steps.
Apart from the rapid methods, CHROMagar™ Salmonella
appeared to be useful for colony isolation, in particular, in highly
contaminated matrices. This medium could conceivably replace
MacConkey agar as the growth agar of choice because it was easier
to read and more quickly differentiated Salmonella from other or-
ganisms. Commercial biochemical assays used by food emergency
response laboratories for confirmation of typical Salmonella col-
onies isolated on selective plating agars such as CHROMagar™
include API 20E, Enterotubes, and Vitek II Compact. One potential
advantage of the automated Vitek II Compact system is that it could
provide higher sample throughput compared to the API 20E, which
was limited to testing smaller sample volumes.
As stated above, diverse and rapid commercial assays for iden-
tifying Salmonella in foods have been evaluated and validated by
food emergency response laboratories. However, most studies have
used artificially spiked samples, which may not truly reflect the
physiological state and acclimation of naturally contaminated
samples. Previous studies have indicated that the currently avail-
able rapid methods might produce a relatively high rate of false
negative results for naturally contaminated samples. Although the
rapid methods require much shorter detection times compared to
conventional methods, some of them still appear to suffer from lack
of sensitivity and specificity for certain sample matrices. Addi-
tionally, the PCR-based detection platforms require extensive
sample preparation steps for some complex matrices and inter-
pretation of the data can be relatively complicated. Finally, as
implied by the outcomes of the studies, participation of the testing
laboratories and their collaborative efforts is crucial to improve the
methods and technologies needed to achieve the goals of inte-
grated laboratory networks and to benefit the laboratories involved
in Salmonella investigations in future.
5. Selection of Salmonella detection methods for rapid
emergency response
Salmonella detection methods to quickly identify Salmonella
contamination sources are vital for immediate actions of emer-
gency responders. Considerable progress has been made to shorten
analysis time of the methods while maintaining or increasing
sensitivity and specificity for detection of Salmonella. However,
despite significant progress in development of a variety of assays to
detect and control Salmonella, the routine use of the assays has
been limited due to some reasons. First of all, the assays have not
been properly selected for sample matrices to be tested and the
methods and samples. Besides, the detection limits of the methods
have not been accurately considered in evaluating the test results.
Another reason may be a long and inappropriate enrichment time.
These limitations are believed to influence the efficacy of the assays
and further mislead the assessment of food safety.
Although the rapid methods are equally efficacious for the
intended use as discussed in the present study, the testing labo-
ratories should consider the following parameters to choose the
most acceptable method, or a combination of the methods, for their
own needs or routine use regardless of the method's principle:
accuracy (specificity and sensitivity), precision, detection limit,
throughput, ease of use, the nature of samples, cost acceptability,
laboratory space, training of laboratory personnel, and quality of
sale services (Blivet et al., 1998; Eijkelkamp et al., 2009; Ivnitski
 K.-M. Lee et al. / Food Control 47 (2015) 264e276
et al., 2000; Mozola, 2006; Tietjen & Fung, 1995). Multiplexing to
simultaneously detect more than one target organism and the
ability to differentiate between naturally contaminated and artifi-
cially spiked samples are also desirable parameters to be included
into consideration in selection of the method. Currently, any single
method may not fulfill all these requirements (Cannons et al., 2006;
Mahon, Murphy, Jones, & Barrow, 1994; Settanni & Corsetti, 2007).
The following parameters are particularly important to be carefully
considered in analytical methods for identification of Salmonella.
5.1. Matrix effect and sample preparation
The efficacy of a rapid method is significantly influenced by
sample matrix (Mozola, 2006; Ricke et al., 1998; Ricci, Volpe,
Micheli, & Palleschi, 2007; Stevens & Jaykus, 2004). Therefore, it
is critical to verify if the method is fitting for the sample matrix to
be tested. Regardless of the detection methods, the sample prepa-
ration is a critical and challenging step that should be optimized to
better separate the target organisms from inhibitory substances in
a sample, which may improve signal to noise ratios and lower false-
positive results (Koyuncu, Andersson, & Ha €ggblom, 2010; Ricke
et al., 1998; Saroj, Shashidhar, Karani, & Bandekar, 2008). Immu-
nomagnetic separation and molecular imprints systems are excel-
lent tools for sample concentration and purification. The rapid
methods that can be used for crude samples and require minimal
sample preparation will be desirable, particularly for field and on-
site usage.
5.2. Selectivity and specificity
The sensitivity and specificity are extensively examined during
the method development and independent studies. However, care
should be given to compare sensitivity and specificity rates of the
methods presented in the published literature and on websites
because the study design and sample matrices used are different
between the studies. It is desirable that both sensitivity and spec-
ificity are as high as possible and can be used to determine whether
a confirmation test will be conducted or not. According to the
previous studies and the manufacturer's claim, the sensitivity and
specificity rates of some commercial assays are more than 99%
(Eijkelkamp et al., 2009). However, such a high level of these
properties may not be achievable by most of commercially available
rapid assays. Even the validation bodies such as AOAC, AFNOR, and
MicroVal do not specify acceptance criteria and values for such
properties. In the meantime, it should point out that the detection
capability of the rapid methods needs to be determined by
considering the bioavailability of toxins and assessment of food
safety, rather than by simply measuring concentrations of patho-
gens by the methods because all Salmonella isolates are not equally
pathogenic for humans.
5.3. Analysis time
In emergency response situations, the rapid methods should be
able to give the results preferably within a few hours to a day and
perform on-site (Skottrup, Nicolaisen, & Justesen, 2008). However,
the commercially available rapid methods which produce the test
results in the next day are rather limited. The presumptive Salmo-
nella results need to be confirmed by culture and biochemical
identification methods, requiring at least additional 24 h and more
for completion. The use of microplates with the rapid methods may
offer the advantage of reducing analysis time and increasing the
capacity of the methods when processing a large number of
samples.
273
5.4. Cost efficiency
The laboratorians seek the rapid methods with lower opera-
tional and maintenance costs with comparable accuracy to avoid
expense and time consuming methods. However, the public health
risk and cost associated with false test results might have much
greater negative impacts than the benefits from the use of a low-
cost method (Eijkelkamp et al., 2009; Tietjen & Fung, 1995).
6. Future aspects of Salmonella detection methods
In order to replace conventional procedures and to meet the
food emergency and safety needs, the rapid methods should be
simple, fast, precise, sensitive, specific, stable, versatile, and cost-
effective. These requirements are in part associated with
improvement of sample preparation and reduced time and labor
for analysis in the methods. According to the literature and previ-
ous studies by food emergency response laboratories on testing and
validating the methods, none of commercially available methods
for detection of Salmonella seem to meet all the requirements for
adequate emergency response to Salmonella incidence.
More reliable and efficient new assays are needed to replace
existing conventional methods and to meet the food emergency
and safety needs although considerable works should be done
before routine use. In addition, the sensitivity and specificity of the
new assays should be more uniformly assessed under a well-
defined study design.
The development and improvement of separation and concen-
tration methods that efficiently isolate, concentrate, and purify
target Salmonella spp. and simultaneously eliminate interfering
components directly from food samples is needed to further in-
crease sensitivity and selectivity of the rapid detection methods
and provide real-time screening of the samples for Salmonella in
significantly less detection time than that required for cultural
enrichments. The removal of the need for cultural enrichment
would allow food emergency response laboratories to detect low
levels of Salmonella contamination in a sample matrix. Further
research needs to focus on the development of separation and
concentration methods to be simple, rapid, low-cost, and appli-
cable to a variety of sample matrices and background microflora.
The development of accurate field and on-site systems may also
help improve the efficiency of food emergency response networks,
reducing analysis time and the work load for reference laboratories.
The progress of microarray and automation technologies will
further increase the capacity of the food emergency response lab-
oratories for screening Salmonella. Since a single Salmonella bac-
terium can be rapidly multiplied into more than a million in a few
hours, the methods should be capable of cultivating extremely low
concentration of the target organisms in samples. The assay which
enables to simultaneously detect multiple pathogens will provide
additional benefits and advantages to the laboratories. Perhaps
more importantly, it is needed to develop the schemes and pro-
cedures to validate the efficiency of new assays in food emergency
response laboratories for coordinating of the results using
dependable and representative control samples.
7. Conclusions
Presently, many commercial detection methods are available
and vary in detection limit, sensitivity, specificity, and cost effi-
ciency with sample matrices to be tested. Despite of great advances
in technologies, current Salmonella detection methods are not fast
and reliable enough for emergency responders to determine the
desired directions within the appropriate period of time, being
required for further improvements and higher efficiency of the
274 K.-M. Lee et al. / Food Control 47 (2015) 264e276
methods. Food emergency response laboratories should choose the
most acceptable method or a combination of methods having the
desired analytical properties to meet food emergency and safety
needs, based on their own needs and requirements. The present
study indicates that validation and comparative studies for the test
kits should be set up under the identical test conditions to better
compare and evaluate the test results among different food emer-
gency response laboratories. A study on the effect of sampling and
sample size on the recovery of the target organism might also help
obtain more accurate test results.
Due to increasing population mobility and the wide distribution
of feed/food products, Salmonella outbreaks and salmonellosis
cases are occurring across state and national boundaries. Such
widely dispersed outbreaks and salmonellosis may limit the public
health resources for detection and investigation of Salmonella
outbreaks. During the past years, some integrated laboratory net-
works have effectively met demands from public and emergency
responders, organized training and the inter-laboratory compara-
tive studies, and coordinated the test results of the participating
laboratories through online reporting and data sharing system.
With years of experience, they should efficiently integrate the
emergency networks for food safety and harmonization of Salmo-
nella detection methods to provide timely investigation of a widely
dispersed multistate Salmonella outbreak and respond to potential
future events of bioterrorism.
Acknowledgments
The authors acknowledge that this study was supported and
made possible by funds from USDA-FSIS Microbiology Cooperative
Agreement Program.
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