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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
2. Conventional Salmonella detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
3. Rapid Salmonella detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
3.1. Immunology-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
3.1.1. Enzyme-linked immunoabsorbent assay (ELISA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
3.1.2. Latex agglutination assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
3.1.3. Immunodiffusion assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
3.1.4. Immunochromatography (dipstick) assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
3.2. Nucleic acid-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
3.2.1. Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
3.2.2. DNA probe hybridization assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
3.3. Miniaturized biochemical assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.4. Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.4.1. Enzyme biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.4.2. Nucleic acid biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.4.3. Antibody-based biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
3.4.4. Micro- and nano-biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

* 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271


5. Selection of Salmonella detection methods for rapid emergency response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
5.1. Matrix effect and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
5.2. Selectivity and specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
5.3. Analysis time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
5.4. Cost efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
6. Future aspects of Salmonella detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

1. Introduction ensure that the results are consistent and comparable among lab-
oratories. Several countries have built regulations and guidelines,
Salmonella bacteria represent the most common and primary such as Regulation (EC) No 2160/2003, the EU Zoonoses Monitoring
cause of food poisoning in many countries for at least over 100 Directive (2003/99/EC), and the Food and Drug Administration
years (Alakomi & Saarela, 2009; Chalker & Blaser, 1988; Coburn, (FDA) Food Code (EC, 2003a, 2003b; FDA, 2009). These regulations
Grassl, & Finlay, 2007). Despite well-established instructions and and guidelines provide or require information on Salmonella and
measures for preventing salmonellosis (Salmonella food poisoning), control methods as well as help develop the food safety rules and
the incidence and severity of human salmonellosis have signifi- regulatory policies, in order to reduce Salmonella pathogens at any
cantly increased. Most salmonellosis cases are self-limiting stage of feed and food production (de Jong & Ekdahl, 2006; Tietjen
(approximately 80%), but large outbreaks caused in schools, hos- & Fung, 1995).
pitals, and restaurants are not very common (Guiney et al., 1995). At Bioterror events, such as anthrax spores in contaminated letters,
present, over 2500 serotypes of Salmonella have been reported have greatly altered the view of the public and the scientific com-
(Fierer & Guiney, 2001). Of these, the most common serotypes munity about bacterial pathogens which could be used as weapons
associated with human illness are Salmonella enterica serovar of biological terrorism, and about the needs and requirements of
typhimurium (S. Typhimurium) and S. enterica serovar Enteritidis the biotechnology for detection of potential bioterrorism-
(S. Enteritidis) in the United States and European countries. employed pathogens (Cannons, Amuso, & Anderson., 2006). Sal-
Approximately, 1.4 million human Salmonella infections occur monella spp. is one of such bacterial pathogen which has been
annually in the United States with assumption of only 2% cases purposely spread mainly using food as a carrier (Ashford et al.,
reported to Center for Disease Control and Prevention (CDC), 2003; To€ro€k et al., 1997). For example, S. Typhimurium was used
resulting in about 16,000 hospitalizations with nearly 600 deaths to infect residents by contaminating salad bars at the local res-
(Cummings et al., 2010; Mead et al., 1999; Turner, 2010). The esti- taurants in Oregon. The United States capability to respond to a
mated total cost associated with Salmonella incidences may be up bioterrorism event has been enhanced by integrated laboratory
to several billion dollars annually (Frenzen et al., 1999; WHO, 2005). networks including the Food Emergency Response Network (FERN)
An estimated annual cost for a Salmonella control program has and Laboratory Response Network (LRN) of Centers for Disease
increased in some countries (WHO, 2005). Much higher incidences Control and Prevention (CDC) as well as to emerging infectious
related to Salmonella may occur in some developing countries diseases.
where relevant data is not readily available. The purpose of this paper is to review Salmonella detection
Salmonella is not considered to be fatal to healthy people or as a methods including detection level, sensitivity, specificity, and
bioweapon agent. However, efforts have been made to develop and sample matrices. Included in the discussion are a summary of Sal-
improve detection technologies for this organism because Salmo- monella testing results and information from food emergency
nella may cause devastating foodborne illness. Salmonella detection response laboratories available in a web-based database portal for
methods are desirable to have sensitivity enough to detect one cell recent fiscal years.
in a defined sample. The analysis time of conventional and rapid
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
Hoorfar, 2010; NordVal, 2009). Such standards are critical to are essentially similar to ISO 6579:2002. The conventional cultural
266 K.-M. Lee et al. / Food Control 47 (2015) 264e276

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
Fig. 1. Scheme of Food Emergency Response Network (FERN)/Food and Drug Admin- sensitivity of serological methods (Hoorfar, Aherns, & Rådstro € m,
istration (FDA) BAM and Salmonella culture method. RV, RappaporteVassiliadis me- 1999; Sørensen, Alban, Nielsen, & Dahl, 2004). If Salmonella iso-
dium; BS, bismuth sulfite agar; HE, Hektoen enteric agar; XLD, xylose lysine
lates cannot be serotyped under certain conditions, PFGE charac-
desoxycholate agar; TT, tetrathionate broth, TSI, Triple sugar iron agar, and LIA, Lysine
iron agar. 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
methods for Salmonella recovery basically include the following the ease of use, reliability of results, high sensitivity and specificity,
essential steps. and lower cost compared to emerging molecular-based technolo-
Pre-enrichment uses a nutritious nonselective medium to gies (Gracias & McKillip, 2004; Maciorowski, Herrera, Jones, Pillai,
recover sublethally injured Salmonella cells while inhibiting the & Ricke, 2006). However, these procedures need to prepare mul-
growth of competing flora (Tietjen & Fung, 1995). Most commonly tiple subcultures required for several identification steps, taking
used media in pre-enrichment step are BPW and lactose broth. The more than 5 days for complete isolation and confirmation. In
pre-enrichment process and formulas have been tested to increase addition, false positive results may occur due to competitive flora
the sensitivity of Salmonella detection. Antimicrobial resistant Sal- (e.g. proteus) (Naravaneni & Jamil, 2005; Swaminathan & Feng,
monella spp. could be highly recovered by using appropriate anti- 1994). Under circumstances in which high throughput screening
biotics in pre-enrichment and selective media (Carrique-Mas & is required for a large number of samples, the laborious and time-
Davies, 2008). The incubated pre-enrichment media are inocu- consuming culture-based techniques may not properly address
lated into selective media containing two or more inhibitory re- such a requirement.
agents such as bile salts, brilliant green, thiosulphate, deoxycholate, The conventional methods have been improved to reduce cost
malachite green, novobiocin, tetrathionate, cycloheximide, nitro- and labor and to offer faster detection and identification of Sal-
furantoin, and sulphacetamide (Arroyo & Arroyo, 1995; Ha, Pillai, & monella. For example, chromogenic and fluorogenic growth media
Ricke, 1995; Ricke, Pillai, Norto, Maciorowski, & Jones, 1998). The (e.g. SM-ID agar, Rambach agar, and BBL CHROMagar Salmonella)
use of inhibitors in a selective media allows continuous growth of used for detection, enumeration, and identification directly on the
Salmonella while suppressing the propagation of other bacteria isolation plate have shown to be convenient, reliable, and more
(Tietjen & Fung, 1995). RappaporteVassiliadis (RV) medium and specific and selective than conventional media (Alakomi & Saarela,
tetrathionate (TT) broth has been used as official Salmonella 2009; Maciorowski et al., 2006; Manafi, 2000; Perry & Freydie re,
enrichment media in approved standard methods such as FDA 2007). The test result using these selective media is typically
Bacteriological Analytical Manual (BAM) and FERN Salmonella available 1 day earlier than conventional methods but is not fast
methods. Enrichment media has been modified to enhance Sal- enough to respond to bioterrorism events, Salmonella outbreak, and
monella growth and thus to increase the sensitivity and selectivity. product recall.
The selective enrichment media containing Salmonella at the
level of above 104 cells ml1 are streaked on solid selective media to 3. Rapid Salmonella detection methods
isolate presumptive positive Salmonella colonies, simultaneously
inhibiting the growth of other bacteria (Carrique-Mas & Davies, Several rapid methods using molecular cloning and recombi-
2008; Ruiz et al., 1996; Tietjen & Fung, 1995). Commonly used nant DNA have been developed, validated, and are available on the
plating media include Salmonella-Shigella agar (SS), brilliant green market. These accelerated Salmonella detection procedures enable
agar (BGA), bismuth-sulfite agar (BSA), Hektoen enteric (HE), and quick answers, reduce storage space of conventional supplies and
xylose-lysine-deoxycholate agar (XLD). The colonies of different samples, and increase the throughput of samples (Alakomi &
Salmonella serotypes are differentiated by colors with the coliforms Saarela, 2009; Blivet, Soumet, Ermel, & Colin, 1998; Tietjen &
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 bioMerieux 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 bioMerieux 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 ) 3.1.1. Enzyme-linked immunoabsorbent assay (ELISA)


Method and Formata Assay Manufacturer Of immunology-based assays, ELISA is the most commonly used
assay for the detection of antigens or products of Salmonella spp.
Sensititre Sensititre
Tri Panel DifcoPasco Lab.
The different ELISA systems have been developed and commer-
Vitek 2 bioMerieux cially available in kit form. In the ELISA assay, an antigen specific to
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 solid matrix. After forming the antigeneantibody complex, the
chain reaction. 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
Fung, 1995). The rapid method may be defined as one which allows infected with Salmonella spp. can be detected using antigens
the detection of Salmonella spp. in samples and delivers reliable coupled to the solid phase of ELISA (Wiuff et al., 2000). ELISAs have
results within a few hours to a day (Ferretti, Mannazzu, Cocolin, also been used to detect antibodies for development of vaccines
Comi, & Clementi, 2001; Tietjen & Fung, 1995). against Salmonella infections (Meenakshi et al., 1999). Several
Commercially available rapid methods for Salmonella detection commercial validated ELISA systems designed for rapid detection of
can be divided into several categories including new selective Salmonella in raw or processed products are available: Assurance
media, modified or adapted conventional procedures, GDS™ for Salmonella (BioControl Systems, Inc., Bellevue, WA),
immunology-based assays, and nucleic acid-based assays (Alakomi TECRA Salmonella (Tecra International Pty Ltd, French Forest, New
& Saarela, 2009; Blivet et al., 1998; Eijkelkamp, Aarts, & van der South Wales, Australia), Salmonella ELISA Test SELECTA/OPTIMA
Fels-Klerx, 2009; Iqbal et al., 2000; Tietjen & Fung, 1995) (Bioline APS, Denmark), and Vitek Immuno Diagnostic Assay Sys-
(Table 1). Of these methods, ELISA and PCR procedures show tem (VIDAS) (BioMerieus, Hazelwood, MO) (Table 1). The aggluti-
comparable specificity and sensitivity to conventional methods. nation technique employs latex particles coated with antibodies
ELISA assays are able to detect Salmonella concentration at the level which react with antigens on the surface of Salmonella cells to form
of 104e105 ml1 while PCR-based assays provide the level of visible aggregates for identification of Salmonella positive samples
sensitivity of 104 ml1 after enrichment. The sensitivity and spec- (Thorns, McLaren, & Sojka, 1994; Tietjen & Fung, 1995). The assays
ificity of these methods largely depend on the background micro- are specific, uncomplicated, and reliable so that generally, they
flora, sample matrix, presence of non-culturable cells, and have been used as a confirmatory analysis technique, rather
inhibitory substances (e.g. fats, proteins, polysaccharides, heavy screening test for Salmonella organisms (Eijkelkamp et al., 2009;
metals, antibiotics, and organic compounds) (Alakomi & Saarela, Love & Sobsey, 2007). There are several commercial kits available
2009; Mozola, 2006; Naravaneni & Jamil, 2005). It has been re- on the market (Table 1).
ported that the sensitivity and detection limits of the methods were
improved by additional sample preparation and purification tech-
3.1.2. Latex agglutination assay
niques such as modification of pre-enrichment and enrichment
The agglutination technique employs latex particles coated with
media, dilution, centrifugation, filtration, flow injection, chroma-
antibodies which react with antigens on the surface of Salmonella
tography, organic solvent extract, and fluorescence hybridization
cells to form visible aggregates for identification of Salmonella
(Kutter, Hartmann, & Schmid, 2006; Mozola, 2006; Polaczyk et al.,
positive samples (Thorns et al., 1994; Tietjen & Fung, 1995). The
2008; Wolffs, Glencross, Thibaudeau, & Griffiths, 2006).
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;
3.1. Immunology-based assays
Love & Sobsey, 2007). There are several commercial kits available
on the market, including Spectate (May & Baker Diagnostics Ltd.,
Immunology-based assays which employ specific mono- or
Glasgow, Scotland, UK), Wellcolex color Salmonella (Wellcolex,
polyclonal antibodies binding with somatic or flagella antigens
Merseyside, UK), Salmonella Latex test (Oxoid, Basingstoke, UK),
have been broadly used for detection of Salmonella spp. in a variety
Bactigen (Wampole Laboratories, Cranbury, NJ), and Slidex (Bio-
of sample matrices (Betts, 2002; Blivet et al., 1998; Iqbal et al.,
Merieux, Marcy L'Etoile, France).
2000; Maciorowski et al., 2006). The immunology-based assays
include enzyme-linked immunoabsorbent assay (ELISA), latex
agglutination tests, immunodiffusion, and immunochromatog- 3.1.3. Immunodiffusion assays
raphy (dipstick). As routine analysis techniques, these assays The Salmonella 1-2 Test system (BioControl Systems, Inc.,
remain a valuable option due to the ability to detect viable non- Bothell, WA) for detection of motile Salmonella is a commercial kit
culturable Salmonella cells (Maciorowski et al., 2006). The previ- that operates on the basis of immunodiffusion reaction (D'Aoust &
ous studies showed that the immunology-based assays are com- Sewell, 1988; Flowers & Klatt, 1989; Nath, Neidert, & Randall, 1989;
parable to and more rapid and specific than cultural methods, Tietjen & Fung, 1995). Before inoculation into the system unit
feasible to isolate Salmonella spp. in conjunction with immuno- which consists of two connected chambers, the sample is pre-
magnetic separation (IMS) techniques to handle large samples, and enriched for 24 h. The enriched sample is inoculated to a tetrathi-
readily automated to reduce time and labor (Keith, 1997; Tietjen & onate brilliant green broth in the inoculation chamber. Salmonella
Fung, 1995; Uyttendaele, Vanwildemeersch, & Debevere, 2003). then moves out of the inoculation chamber into the mobility
However, these methods have some potential shortcomings for chamber in which antibody has been added onto a distal surface of
detection of Salmonella including a longer enrichment time to a semisolid medium. Salmonella in the mobility chamber is
obtain the appropriate number of cells, cross-reactions with closely immobilized by forming an antigeneantibody complex. After in-
related antigens, antigen variation, limits in sensitivity for some cubation for 14 h, the readable three-dimensional immunodiffusion
sample matrices and stressed cells, and high cost for automation band is produced. Modifications in an enrichment step before
(Blivet et al., 1998; Bolton, Fritz, & Poynton, 2000; Dill, Stanker, & inoculation and an increase of incubation time improved the
Young, 1999; Uyttendaele et al., 2003). effectiveness of detection of Salmonella spp. (Nath et al., 1989). The
K.-M. Lee et al. / Food Control 47 (2015) 264e276 269

modified and original assays have shown to be equivalent with the Warrington, UK), Probelia (Sanofi -Diagnostics Pasteur, Marnes-la-
reference culture method. Coquette, France), BAX system (DuPont Qualicon, Wilmington, DE),
TagMan (PE-Applied Biosystems, Foster City, CA), Gene-Trak (Neo-
3.1.4. Immunochromatography (dipstick) assays gen Corporation, Lansing, MI), iQ-Check™ PCR (BioRad Labora-
The commercial Salmonella test kits, based on the dipstick tories, Hercules, CA), LightCycler (Roche Diagnostics, Manheim,
format, include the Tecra® Salmonella Unique™ test (Tecra Inter- Germany), and SmartCycler (Cepheid Inc., Sunnyville, CA). The BAX
national Pty Ltd, French Forest, New South Wales, Australia) and the system is the first commercially available PCR method using a
PATH-STIK (Celsis, Inc., Edison, NJ). The Tecra Salmonella Unique™ single tablet combining all reagents (primers, enzyme, and de-
test which consists of the immunoenrichment and detection steps oxyribonucleotides) needed for PCR to rapidly amplify and detect
is a screening procedure for Salmonella detection. The PATH-STIK Salmonella spp. (Bennett,., Greenwood, Tennant, Banks, & Betts,
Salmonella test is an immunological dipstick assay for one-step 1998; Hoorfar et al., 1999; Maciorowski et al., 2006).
detection of Salmonella. The dipstick contains an assay control The real-time PCR (RT-PCR) system detects accumulated PCR
and all reagents used for Salmonella detection. Prior to the dipstick product by monitoring the increase fluorescence signal using the
assay, a sample is pre-enriched and selectively enriched in media. integrated real-time thermocycler and fluorescence detector
The enriched sample is applied to the test unit and Salmonella in the within a system. This helped overcome the problem of false positive
sample is captured onto the dipstick. Unlike the Tecra® Salmonella results posed by amplicon contamination (Maurer, 2011). The false
Unique™ test, the PATH-STIK doesn't include a washing step, positive results can be further excluded by an internal standard
requiring only 30 min for analysis (van Beurden, 1992; Brinkman, used for ensuring the absence of inhibitors. The RT-PCR system
van Beurden, Mackintosh, & Beumer, 1995). employing automated DNA extraction, amplification, and detection
has improved the ability of industry and emergency response lab-
3.2. Nucleic acid-based assays oratories in identifying Salmonella from a variety of sample
matrices. The RT-PCR methods available on the market can be
The nucleic acid-based assays are Salmonella detection tests that classified into several categories based on the principle and
utilize a specific nucleic acid target sequence within the organism. approach of the methods. Several fluorescent techniques have been
The assays have been most intensively explored and developed for developed and incorporated into the RT-PCR systems for simplicity
the past decade among Salmonella detection methods because they and cost saving in detecting the target Salmonella sequences.
offer some advantages of sensitivity, specificity, and inclusivity over
other methods, rapidly identifying Salmonella without obtaining 3.2.2. DNA probe hybridization assay
pure cultures (Glynn et al., 2006; Mozola, 2006). Two major tech- A DNA probe hybridization assay uses a DNA probe with a
niques of the assays are direct hybridization (DNA probe) and sequence complementary to the target sequence of a DNA or RNA
amplification (PCR) methods. The great progress of the assays al- molecule in the target organism (de Boer & Beumer, 1999; Fung,
lows the detection of very low numbers of organisms in the sample 2002; Mozola, 2006). In this assay, the target cells are first lysed
and high throughput of a large number of samples for routine and the produced nucleic acids are purified prior to hybridization
analysis (Cohen et al., 1993; Mozola, 2006; Rasmussen, Rasmussen, with a DNA probe. The unbound probe to a target sequence is
Larsen, Hoff-Jorgensen, & Cano, 1994). eliminated by washing. The stable hybrid with the labeled DNA can
be detected using different detection techniques, such as radio-
3.2.1. Polymerase chain reaction (PCR) isotopes and enzymatic reactions. The presumptive Salmonella re-
The PCR assay is the preferred non-cultural technique for in vitro sults are typically confirmed by the culture methods.
primer-meditated enzymatic amplification of specific segments of The DNA probe hybridization assays have some advantages in
DNA for the detection of Salmonella pathogens (Cocolin, Manzano, specificity and sensitivity of identifying Salmonella in samples
Cantoni, & Comi, 1998; McKillip & Drake, 2004; Wolcott, 1991). The compared to other methods. The assays are effective to rapidly screen
PCR assay can exponentially amplify a single specific sequence to the large numbers of samples and highly specific to detect the target
one million-fold of DNA fragment within 2 or 3 h using a ther- cells without cross-reactions with the other organism (Agron et al.,
mostable DNA polymerase. The amplified products can be detected 2001; de Boer & Beumer, 1999; Dunbar & Jacobson, 2007; Mozola,
by several means (e.g. gel-based systems and real-time PCR) 2006). This may allow more rapid distribution of Salmonella-free
(Eijkelkamp et al., 2009; Mozola, 2006). The development and products and more frequent monitoring at important investigation
advancement of the technique for amplifying specific segment of sites. However, the required sensitivity may be achieved only in the
DNA improve the specificity and sensitivity enough for detecting presence of sufficient concentration of target organisms after pre-
even one molecule of target DNA in a defined sample. Due to the enrichment (Jones, Law, & Bej, 1993). The amplification of target
capability to detect such a low concentration of Salmonella, nucleic acid sequence and DNA probe might be an alternative way to
enrichment times are considerably shorter to reach Salmonella increase the sensitivity of the assays (Tietjen & Fung, 1995).
concentration needed for reliable detection by the PCR compared to The Gene-Trak® Salmonella Assay (Neogen Corporation, Lansing,
other assays. The equivalency was reported between the PCR assay MI) is the first introduced commercial assay. The second generation
and the standard culture methods in detecting Salmonella in food of this assay is based on a hybridization format of two probes, a
products (Aabo, Andersen, & Olsen, 1995; Cano, Rasmussen, polyadenylic acid (poly dA) tail on the capture probe and a fluo-
Sanchez Fraga, & Palomares, 1993; Eyigor, Carli, & Unal, 2002; rescein labeled detector probe, to the Salmonella ribosomal RNA
Stone, Oberst, Hays, McVey, & Chengappa, 1994). The PCR assays (rRNA) and enzyme-mediated colorimetric detection. The Gene-
have been developed, validated, and standardized according to the Trak® Salmonella Assay has been modified to the direct-labeled
general guidelines and requirements by ISO (IOS 20838:2006, ISO/ format which replaces a fluorescein labeled detector probe with
DIS 22119, ISO 16140:2003, ISO 6579:2002, ISO 22174). HRP. This assay is equivalent to the original dipstick format assay
Commercial kits based on the PCR technology have been and greatly simplifies the procedure by eliminating the enzyme
developed, and successfully used for routine Salmonella screening incubation and washing steps and by lowering a hybridization
in the industry and emergency response laboratories. The PCR- temperature (Eijkelkamp et al., 2009; Mozola, 2006). The microwell
based commercial kits and systems with different specificity and format assay was also introduced and available on the market un-
sensitivity include ABI Prism 7500 (Applied Biosystems, 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 previous studies reported that miniaturized biochemical
the market have less than 48 h of a test time frame. Since the assays systems are accurate, efficient, convenient, labor-saving, space-
require higher detection limits of enrichment culture for positive saving, and more economical compared to the conventional culture
signal, they need a longer enrichment time to increase the con- methods (Cox, Fung, Bailey, Hartman, & Vasavada, 1987; Feng,
centration of Salmonella in the final culture. However, the total test 1997; Holmes, 1989; Kalamaki, Prioce, & Fung, 1997; Stager &
time with certain samples can be shortened to a significant extent. Davis, 1992). Except for the MICRO-ID and APE 20E system
GeneQuence® Salmonella showed a sensitivity of 97.1% using a new requiring 4 h of incubation, the results are typically obtained after
2-stage procedures including 24e27 h pre-enrichment and selec- 24 h of incubation at an appropriate temperature in other systems.
tive enrichment followed by a 2 h testing time in selected foods These miniaturized biochemical systems and kits have significantly
(Alles, Peng, Wendorf, & Mozola, 2007). The assays based on such contributed to improve accuracy, efficiency, and capacity in
rRNA hybridization approach have certain benefits in designing a detecting Salmonella spp. It is anticipated that in the future they
probe because rRNA sequences are highly conserved, stable, and will continue to play an important role in the food, industrial, or
abundant in a bacteria cell, providing various targets and increasing environmental microbiology areas.
the assay sensitivity (Mozola, Peng, & Wendorf, 2007).
3.4. Biosensors
3.3. Miniaturized biochemical assay
Biosensor technology encompasses an applied microbiology
Miniaturized biochemical assay employs the reduced volumes approach to bacterial detection (Alonso-Lomillo, Domínguez-
of reagents, media, and vessels for rapid characterization of Sal- Renedo, & Arcos-Martínez, 2010; Baeumner, 2003; Fung, 2002;
monella to replace conventional biochemical tests and to provide Lazcka, Campo, & Mun  oz, 2007) that defines a molecule or a
more results in the same span of time (Fung, 2002; Ge & Meng, group of molecules by incorporating materials immobilized on the
2009; Tietjen & Fung, 1995). Many microbiologists have devel- surface of a physicochemical transducer or transducing micro-
oped and used diverse miniaturized biochemical systems to rapidly system. A recognition signal is generated when a specific analyte
confirm isolated organisms from a larger number of samples. Most binds to the biological recognition element. The signal can be
miniaturized biochemical systems consist of disposable sterilized changes in mass, oxygen consumption, potential difference,
microtiter plates, a multiple inoculation device, and 10e20 media refractive index, pH, current, and other parameters (Baeumner,
or substrates specially designed to target organisms. Salmonella 2003; Eijkelkamp et al., 2009). It is anticipated that a biosensor
spp. are identified based on the assimilation and utilization of technique may replace existing immunology- and nucleic acid-
special substrate and automated colony and cell density measure- based assays (Alocilja & Radke, 2003; Lazcka et al., 2007; Van
ment. The test procedures include placing pure cultures into wells Dorst et al., 2010).
of a microtiter plate each of which represents independent inocu- Biological recognition elements used in biosensor application
lation in the conventional method, holding up to 96 different cul- include enzymes, antibodies, nucleic acids, whole cells, tissue/
tures. After 16e24 h incubation at a desirable temperature, the whole organisms, and biomimetic material. The signal recognition
presence or absence of Salmonella on solid media or liquid media and transduction in the biosensor is achieved by different types of
can be determined by reading color changes caused by the reaction transducers: electrochemical, optical, thermometric, micro-
of specific compositions and metabolites with reagents with the aid mechanical, and other miscellaneous transducers according to
of a manual identification code or an automatic reader interfaced which the biosensors have been often classified by several authors
with a computer. (Goepel & Heiduschka, 1995; Ivnitski, Abdel-Hamid, Atanasov,
Diverse miniaturized kits for rapid biochemical characteriza- Wilkins, & Stricker, 2000; Leonard et al., 2003).
tion of Salmonella are commercially available, including API 20E
(bioMe rieux sa, Marcy-I'Etoile, France), Enterotube II (BD Di- 3.4.1. Enzyme biosensor
agnostics, Sparks, MD), Enterobacteriaceae Set II (previously An enzyme biosensor is an analytical device that integrates an
known as Mnitek II, BBL Microbiology Systems, Cockeysville, MD), enzyme with a transducer to produce a signal resulting from
MICRO-ID (Remel, Lenexa, KS), EPS (Enteric Pathogens Screen different physicochemical changes caused by enzyme-catalyzed
Card) (Vitek Systems, Hazelwood, MO), GNI (Gram Negative reactions. The signal is converted into measurable responses such
Identification Card) (Vitek Systems, Hazelwood, MO), Microscan as current, temperature change, and light absorption to determine
(Baxter Diagnostics, West Sacramento, CA), Quad Enteric Panel target analyte concentration (Baeumner, 2003). Enzyme biosensors
(Micro-Media Systems, San Jose, CA), Quantum II (Abbott Labo- are highly selective, rapid, and easy to use while they are expensive
ratories, Diagnostic Division, Abbott Park, IL), Sensititre (Sensititre, and sometimes lose their activities when immobilized on a trans-
Salem, NH), Tri Panel (DifcoPasco Laboratories, Wheat Ridge, CA), ducer. Enzymes used in this type of biosensor include glucose ox-
Vitek 2 (bioMe rieux sa, Marcy-I'Etoile, France), and Walk Away idase, galactosidase, glucoamlyase, lactate oxidase, and other
(Dade MicroScan, West Sacramento, CA). The majority of these enzymes (Fung, 2002).
commercial kits were first used for comparative analyses of clin-
ical samples and later applied for food microbiological samples 3.4.2. Nucleic acid biosensor
(Fung, 2002; Stager & Davis, 1992). These systems use similar A nucleic acid biosensor which consists of a nucleic acid as a
approaches and procedures ideal for analyzing large number of biological recognition element and a signal transducer is based on
samples and isolates. The sensitivity and specificity of these sys- the hybridization of complementary strands of DNA or RNA mole-
tems, particularly the latest ones, easily exceed 95% with the cules for detection of organisms (Baeumner, 2003; Eijkelkamp
higher sample throughput, but the analysis cost may significantly et al., 2009; Lazcka et al., 2007). In addition to detection of bacte-
increase if a sophisticated automated identification system is used. ria and other pathogens, nucleic acid biosensors have been applied
Most miniaturized biochemical assay kits were initially designed to food and environmental samples for detection of toxic com-
for identification of gram-negative bacteria including Salmonella, pounds and biotechnology products using different signal trans-
Shigella, and Enterobacteriaceae, and later expanded to gram duction systems such as quartz crystal microbalances, surface
positive bacteria, anaerobes, and even yeasts and molds (Gracias & plasmon resonance, surface acoustic wave sensor, and evanescent
McKillip, 2004). wave biosensors (Deisingh & Thompson, 2004; Feriotto, Borgatti,
K.-M. Lee et al. / Food Control 47 (2015) 264e276 271

Mischiati, Bianchi, & Gambari, 2002; Mascini, Palchetti, & detectability and concentration. Furthermore, wild-type Salmonella
Marrazza, 2001). enteriditis showed much lower recoveries from liquid egg products
relative to the American Type Culture Collection (ATCC) strains.
3.4.3. Antibody-based biosensors In comparison studies of recovery of Salmonella from culture
Antibody-based biosensors are analytical devices which use an media, Yersinia pestis enrichment (YPE) broth and buffered peptone
antibody as a biological recognition element attached to a signal water (BPW) were as effective as lactose broth, a standard enrich-
transducer to quantify the target analyte concentration (Bilitewski, ment medium, in some sample matrices. Overall, xylose lysine
2000; Rogers, 2000). Antibodies are the most commonly used deoxycholate (XLD) agar exhibited a higher recovery of Salmonella
biological recognition element due to their high specificity and spp. than Hektoen enteric (HE) and bismuth sulfite (BS) agars,
affinity to antigen, diversity, and availability on the market. which may be explained by the selectivity of the media and the
Antibody-based biosensors can be divided into three main assay physiological properties of the strains used in the studies. Sample
formats including an immobilized antibody, immobilized antigen, preparation techniques also appeared to affect the recovery of the
and non-immobilized antibody or antigen whose characteristics spiked samples. For example, results obtained with the VIDAS® SLM
are largely determined by the type of signal transduction mecha- and BAX® Q7 Salmonella PCR assays indicated that washing with
nisms (Rogers, 2000). soaking and soaking alone were more effective for extraction of
Salmonella than surface washing only.
3.4.4. Micro- and nano-biosensors Comparative and collaborative studies of the rapid methods for
The field of micro- and nano-biosensors has been drawing much screening of Salmonella have been performed by many food
attention from microbiologist due to their great potential to solve emergency response laboratories by using a variety of spiked
many current problems including real time detection of a variety of sample matrices. However, as seen in Table 2, only limited numbers
pathogens, and believed as one of the future directions to detect of methods and platforms were extensively evaluated and
Salmonella spp. (Chen et al., 2005; Mozola, 2006; Scaria et al., compared within integrated laboratory networks during this spe-
2008). However, actual application to Salmonella detection is cific period. In the validation studies, a suspension of Salmonella
rather limited so far because of the instrumental complexity and cells at the appropriate turbidity was serially diluted for spiking
immature stage of the technology development (Alakomi & into samples prior to assessing the test kits. Enrichment using BPW
Saarela, 2009). However, the techniques are rather restricted in or YPE broth for the PCR-based methods offered a rapid alternative
application to pathogen detection because of their incompatibility to the standard culture method for identifying Salmonella spp. Of
with some biological samples. Despite some obstacles and chal- the PCR methods tested, the BAX™ system showed comparable
lenges in the research and application of the micro- and nano- sensitivity and specificity to conventional culture methods in most
biosensors, rapid development and integration of associated core sample matrices spiked at low and high concentrations of Salmo-
technologies seems to provide no doubt that in the future they nella cells, with the exception of some false results due to cross-
would be one of important technologies in microbiology and have a contamination of PCR products. No differences in identifying Sal-
great value in the market. monella spp. were found between the standard BAX™ system and
the BAX™ Q7 system.
4. Salmonella detection methods for food emergency Overall, there was little or no difference between the rapid
response laboratories methods, which displayed equally high accuracies in identification
of the target organisms regardless of the matrix. Comparable re-
Salmonella detection is critical for emergency responders who sults were found between BAX and SmartCycler PCR platforms in
require robust and preferably transportable laboratory-based single laboratory testing. Some laboratories reported that the per-
detection systems. The methods for Salmonella detection typically formance and accuracy of the SmartCycler PCR system was some-
recommended by the FDA and the FSIS of the USDA are based on what dependent on the characteristics of the sample matrix. It was
cultural, serological, and biochemical properties using selective also reported that the sensitivity of the systems was further
media. However, rapid methods for Salmonella detection have improved by increasing enrichment time. Taken together, these
become increasingly important for effectively monitoring food observations seem to imply that the difference between these
products and are considered desirable as a future approach and methods may be mainly due to the matrix properties and the
national strategy. Rapid test kits recommended by the US Food possible presence of PCR inhibitors, rather than the inherent
Emergency Response Network for Salmonella include the VIDAS effectiveness of the assay systems.
Salmonella (SLM) and immuno-concentration Salmonella (ICS) Several laboratories have compared the BAX™ Q7 PCR and
methods, the Tecra Unique Salmonella test, and the polymerase VIDAS Salmonella (SLM) assays. The results showed no significant
chain reaction (PCR)-based BAX™ system, which were chosen with difference between the two assays in detecting Salmonella spp. in
the expectation of providing results within 24 h. These test kits are most food matrices. Indeed, most failures of the two assays in
approved for screening purposes by the AOAC and have been Salmonella detection appeared to be related to the matrix effect.
widely used by food testing laboratories. Several laboratories have indicated that the VIDAS assay might be
During the past four fiscal years, food emergency response more reliable than the BAX system based on the study results and
laboratories have performed single lab and/or multi-lab validations on the discussions moderated through a web-based database por-
involving rapid Salmonella detection platforms. Several studies tal. However, the VIDAS assay was insufficiently sensitive for
have focused on the recovery of Salmonella spp. from a variety of certain matrices (e.g., bean sprouts), and requires a secondary
sample matrices spiked with Salmonella strains. According to the enrichment procedure to reduce the effects of background flora.
results of these studies, detection levels varied with sample matrix Furthermore, there is only limited information available on how the
and spiking concentration. For example, when spiked at the same BAX™ and VIDAS systems interact with matrices.
low concentration Salmonella was easily identified in cat food and Several immunomagnetic separation (IMS) systems such as the
vegetable burgers, while its detection in other matrices such as Dynal BeadRetriever, Pathatrix, and BioVeris M1M Platform were
baby oatmeal, peanut butter, liquid eggs, and cantaloupe was more validated and assessed for their efficiency in recovering and
difficult. Several food matrices, including milk, strawberries, and isolating different strains of Salmonella from sample matrices
creamed corn, displayed an inverse relationship between (Table 2). Typically, after an overnight incubation, a sample aliquot
272 K.-M. Lee et al. / Food Control 47 (2015) 264e276

Table 2 that the system efficiency is highly dependent on sample matrix. In


Salmonella detection methods for screening of Salmonella and sample matrices used addition, the IMS systems appeared to be not fully functional when
for the studies in food emergency response laboratories during the past fiscal years.
the complexity of the sample matrices increased. Compared to
Methoda Sample matrix manual methods, the automated IMS systems further increased the
PCR assays speed and productivity, and minimized cross-contamination by
ABI 7500 Fast Cheese, cookie, cookie, ground beef, ice significantly reducing sample transfer time and pipetting steps.
cream, liquid egg, peanut butter, potato Apart from the rapid methods, CHROMagar™ Salmonella
salad, cracker, snack bar
appeared to be useful for colony isolation, in particular, in highly
BAX® PCR system Baby carrot, beef stick, black pepper,
cilantro, cilantro flakes-dried, cooked contaminated matrices. This medium could conceivably replace
ham, cooked ham, deli ham, ground MacConkey agar as the growth agar of choice because it was easier
beef, ground beef, hamburger, hot to read and more quickly differentiated Salmonella from other or-
pepper, hot dog, infant formula,
ganisms. Commercial biochemical assays used by food emergency
jalapeno pepper, liquid egg, milk,
orange juice, peanut butter, potato
response laboratories for confirmation of typical Salmonella col-
salad, powdered formula, precut ready- onies isolated on selective plating agars such as CHROMagar™
to-eat (RTE) lettuce, serrano pepper, include API 20E, Enterotubes, and Vitek II Compact. One potential
spinach, sponge sicle, tomato, vegetable advantage of the automated Vitek II Compact system is that it could
juice, whole head romaine lettuce
provide higher sample throughput compared to the API 20E, which
LightCycler Alfalfa sprout, baby oatmeal, broccoli,
cantaloupe, cat food, chicken, creamed was limited to testing smaller sample volumes.
corn, frozen spinach, ground beef, hot As stated above, diverse and rapid commercial assays for iden-
dog, jalapeno pepper, lettuce, liquid tifying Salmonella in foods have been evaluated and validated by
egg, milk, orange juice, peanut butter, food emergency response laboratories. However, most studies have
strawberry, tomato, vegetable burger,
watermelon
used artificially spiked samples, which may not truly reflect the
SmartCycler Baby carrot, cheese, chicken breast deli physiological state and acclimation of naturally contaminated
meat, cookie, deli ham, ground beef, ice samples. Previous studies have indicated that the currently avail-
cream, liquid egg, milk, orange juice, able rapid methods might produce a relatively high rate of false
peanut butter, potato salad, raisin, raw
negative results for naturally contaminated samples. Although the
pork sausage, cracker, snack bar,
spinach, vegetable juice rapid methods require much shorter detection times compared to
Luminex Alfalfa sprout, chocolate, bean sprout conventional methods, some of them still appear to suffer from lack
ELISA assay of sensitivity and specificity for certain sample matrices. Addi-
VIDAS Cilantro, cilantro flakes-dried, jalapeno tionally, the PCR-based detection platforms require extensive
pepper, precut ready-to-eat (RTE)
lettuce, serrano pepper, spinach, whole
sample preparation steps for some complex matrices and inter-
head romaine lettuce pretation of the data can be relatively complicated. Finally, as
IMS assays implied by the outcomes of the studies, participation of the testing
Dynal BeadRetriever Chicken breast deli meat, cooked ham, laboratories and their collaborative efforts is crucial to improve the
ground beef, hamburger, liquid egg,
methods and technologies needed to achieve the goals of inte-
milk, orange juice, peanut butter, potato
salad, raisin, raw pork sausage, spinach grated laboratory networks and to benefit the laboratories involved
M1M analyzer Alfalfa sprout, baby oatmeal, broccoli, in Salmonella investigations in future.
cantaloupe, cat food, chicken, creamed
corn, frozen spinach, ground beef, hot 5. Selection of Salmonella detection methods for rapid
dog, jalapeno pepper, lettuce, liquid
egg, milk, orange juice, peanut butter,
emergency response
strawberry, tomato, vegetable burger,
watermelon Salmonella detection methods to quickly identify Salmonella
PATHATRIX Hot pepper, peanut butter, tomato contamination sources are vital for immediate actions of emer-
Miniaturized biochemical assays
gency responders. Considerable progress has been made to shorten
API 20E Chicken breast deli meat, raisin, raw
pork sausage analysis time of the methods while maintaining or increasing
Enterotubes Liquid egg, milk, orange juice, peanut sensitivity and specificity for detection of Salmonella. However,
butter, spinach despite significant progress in development of a variety of assays to
Vitek® Black pepper, ground beef, peanut detect and control Salmonella, the routine use of the assays has
butter, potato salad, powdered formula
Media
been limited due to some reasons. First of all, the assays have not
CHROMagar Ground beef, liquid egg, potato salad been properly selected for sample matrices to be tested and the
a sampling and sample preparation have not been optimized for the
PCR, Polymerase chain reaction; ELISA, enzyme-linked immunoabsorbent assay;
IMS, immunomagnetic separation. 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
was subjected to immunomagnetic separation and the two bacte- and further mislead the assessment of food safety.
rial fractions (immuno-bound and unbound) were plated onto Although the rapid methods are equally efficacious for the
agarified medium and/or processed using the Roche LightCycler 2.0 intended use as discussed in the present study, the testing labo-
RT-PCR, BAX System Q7 PCR, or Applied Biosystems 7500. Irre- ratories should consider the following parameters to choose the
spective of manufacturer and concentration of Salmonella, the IMS most acceptable method, or a combination of the methods, for their
systems were generally effective for improving the recovery from own needs or routine use regardless of the method's principle:
sample matrices and allowing for shorter turnaround times. accuracy (specificity and sensitivity), precision, detection limit,
However, the recovery from some food matrices (e.g., bean sprouts, throughput, ease of use, the nature of samples, cost acceptability,
cooked ham, ground beef, and peanut butter) was identical with or laboratory space, training of laboratory personnel, and quality of
without the use of an IMS system at all spiking levels, indicating sale services (Blivet et al., 1998; Eijkelkamp et al., 2009; Ivnitski
K.-M. Lee et al. / Food Control 47 (2015) 264e276 273

et al., 2000; Mozola, 2006; Tietjen & Fung, 1995). Multiplexing to 5.4. Cost efficiency
simultaneously detect more than one target organism and the
ability to differentiate between naturally contaminated and artifi- The laboratorians seek the rapid methods with lower opera-
cially spiked samples are also desirable parameters to be included tional and maintenance costs with comparable accuracy to avoid
into consideration in selection of the method. Currently, any single expense and time consuming methods. However, the public health
method may not fulfill all these requirements (Cannons et al., 2006; risk and cost associated with false test results might have much
Mahon, Murphy, Jones, & Barrow, 1994; Settanni & Corsetti, 2007). greater negative impacts than the benefits from the use of a low-
The following parameters are particularly important to be carefully cost method (Eijkelkamp et al., 2009; Tietjen & Fung, 1995).
considered in analytical methods for identification of Salmonella.
6. Future aspects of Salmonella detection methods
5.1. Matrix effect and sample preparation
In order to replace conventional procedures and to meet the
The efficacy of a rapid method is significantly influenced by food emergency and safety needs, the rapid methods should be
sample matrix (Mozola, 2006; Ricke et al., 1998; Ricci, Volpe, simple, fast, precise, sensitive, specific, stable, versatile, and cost-
Micheli, & Palleschi, 2007; Stevens & Jaykus, 2004). Therefore, it effective. These requirements are in part associated with
is critical to verify if the method is fitting for the sample matrix to improvement of sample preparation and reduced time and labor
be tested. Regardless of the detection methods, the sample prepa- for analysis in the methods. According to the literature and previ-
ration is a critical and challenging step that should be optimized to ous studies by food emergency response laboratories on testing and
better separate the target organisms from inhibitory substances in validating the methods, none of commercially available methods
a sample, which may improve signal to noise ratios and lower false- for detection of Salmonella seem to meet all the requirements for
positive results (Koyuncu, Andersson, & Ha €ggblom, 2010; Ricke adequate emergency response to Salmonella incidence.
et al., 1998; Saroj, Shashidhar, Karani, & Bandekar, 2008). Immu- More reliable and efficient new assays are needed to replace
nomagnetic separation and molecular imprints systems are excel- existing conventional methods and to meet the food emergency
lent tools for sample concentration and purification. The rapid and safety needs although considerable works should be done
methods that can be used for crude samples and require minimal before routine use. In addition, the sensitivity and specificity of the
sample preparation will be desirable, particularly for field and on- new assays should be more uniformly assessed under a well-
site usage. defined study design.
The development and improvement of separation and concen-
tration methods that efficiently isolate, concentrate, and purify
5.2. Selectivity and specificity target Salmonella spp. and simultaneously eliminate interfering
components directly from food samples is needed to further in-
The sensitivity and specificity are extensively examined during crease sensitivity and selectivity of the rapid detection methods
the method development and independent studies. However, care and provide real-time screening of the samples for Salmonella in
should be given to compare sensitivity and specificity rates of the significantly less detection time than that required for cultural
methods presented in the published literature and on websites enrichments. The removal of the need for cultural enrichment
because the study design and sample matrices used are different would allow food emergency response laboratories to detect low
between the studies. It is desirable that both sensitivity and spec- levels of Salmonella contamination in a sample matrix. Further
ificity are as high as possible and can be used to determine whether research needs to focus on the development of separation and
a confirmation test will be conducted or not. According to the concentration methods to be simple, rapid, low-cost, and appli-
previous studies and the manufacturer's claim, the sensitivity and cable to a variety of sample matrices and background microflora.
specificity rates of some commercial assays are more than 99% The development of accurate field and on-site systems may also
(Eijkelkamp et al., 2009). However, such a high level of these help improve the efficiency of food emergency response networks,
properties may not be achievable by most of commercially available reducing analysis time and the work load for reference laboratories.
rapid assays. Even the validation bodies such as AOAC, AFNOR, and The progress of microarray and automation technologies will
MicroVal do not specify acceptance criteria and values for such further increase the capacity of the food emergency response lab-
properties. In the meantime, it should point out that the detection oratories for screening Salmonella. Since a single Salmonella bac-
capability of the rapid methods needs to be determined by terium can be rapidly multiplied into more than a million in a few
considering the bioavailability of toxins and assessment of food hours, the methods should be capable of cultivating extremely low
safety, rather than by simply measuring concentrations of patho- concentration of the target organisms in samples. The assay which
gens by the methods because all Salmonella isolates are not equally enables to simultaneously detect multiple pathogens will provide
pathogenic for humans. additional benefits and advantages to the laboratories. Perhaps
more importantly, it is needed to develop the schemes and pro-
5.3. Analysis time cedures to validate the efficiency of new assays in food emergency
response laboratories for coordinating of the results using
In emergency response situations, the rapid methods should be dependable and representative control samples.
able to give the results preferably within a few hours to a day and
perform on-site (Skottrup, Nicolaisen, & Justesen, 2008). However, 7. Conclusions
the commercially available rapid methods which produce the test
results in the next day are rather limited. The presumptive Salmo- Presently, many commercial detection methods are available
nella results need to be confirmed by culture and biochemical and vary in detection limit, sensitivity, specificity, and cost effi-
identification methods, requiring at least additional 24 h and more ciency with sample matrices to be tested. Despite of great advances
for completion. The use of microplates with the rapid methods may in technologies, current Salmonella detection methods are not fast
offer the advantage of reducing analysis time and increasing the and reliable enough for emergency responders to determine the
capacity of the methods when processing a large number of desired directions within the appropriate period of time, being
samples. required for further improvements and higher efficiency of the
274 K.-M. Lee et al. / Food Control 47 (2015) 264e276

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