Innovative diagnostic methods were developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in
minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and
evaluate their efficacy and viability for routine analysis. To this purpose, ready-to-eat fresh fruits and vegetables were collected throughout the
production chain. A multidisciplinary approach, including a newly developed ELISA method compared to ISO procedures, was applied to detect the
pathogenic bacteria after harvesting, processing and shelf-life. Results obtained exhibited the technical efficiency of the developed methods showing
similar sensitivity, specificity, negative predictive values and negative likelihood ratios

A. Ferrante

A. Hadjilouka

E.H. Drosinos

M. Cavaiuolo

P. Tzamalis

S. Paramithiotis

AIR Universita degli studi di Milano

     98 Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014
                                   www.world-food.net                            Journal of Food, Agriculture & Environment Vol.12 (3&4): 98-99. 2014
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Validation of ELISA-based detection of L. monocytogenes and E. coli O157:H7 in
fresh cut vegetables
Marina Cavaiuolo 1, Antonio Ferrante 1*, Spiros Paramithiotis 2, Agni Hadjilouka 2, Periklis Tzamalis 2
and Eleftherios H. Drosinos 2
1 Dept. Agricultural and Environmental Sciences, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy.
2 Laboratory of Food Quality Control and Hygiene, Department of Food Science and Human Nutrition, Agricultural University
of Athens, Iera Odos 75, GR-118 55 Athens, Greece.
e-mails: marina.cavaiuolo@unimi.it, antonio.ferrante@unimi.it, sdp@aua.gr, agni_xatz@aua.gr, pertzam@otenet.gr,ehd@aua.gr
Received 12 June 2014, accepted 20 September 2014.
Abstract
Innovative diagnostic methods were developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in
minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and
evaluate their efficacy and viability for routine analysis. To this purpose, ready-to-eat fresh fruits and vegetables were collected throughout the
production chain. A multidisciplinary approach, including a newly developed ELISA method compared to ISO procedures, was applied to detect the
pathogenic bacteria after harvesting, processing and shelf-life. Results obtained exhibited the technical efficiency of the developed methods showing
similar sensitivity, specificity, negative predictive values and negative likelihood ratios.
Key words: Leafy vegetables, melon, vegetables, ELISA, ISO.
Introduction
Ready to eat fresh-cut vegetables (RTEs) are convenient foods
that have increased the volume and value of commercialisation
among different European countries. Nevertheless, the economic
crisis in the recent years has slightly affected the fresh-cut fruits
and vegetables market. This trend can be explained considering
the higher quality of the products, which must be convenient and
safe 1. Most of the leafy vegetables used for the fresh-cut
preparation are grown in the soil and bacteria contamination can
easily occur. Moreover, their short growing cycles require an
higher supply of organic matter in order to keep the soil fertility.
The organic matter supply is usually performed by manure and as
such represents a possible source of contamination of human
pathogenic bacteria like Escherichia coli O157 L. and Listeria
monocytogenes L. 2.
Washing treatments are able to reduce the bacteria load and
allow to preserve the RTE products for long time 3. The official
standard procedures for the detection of bacterial pathogens
require 4-5 days. Unfortunately, the shelf life of leafy vegetable
products is limited to 5-7 days that include two days of processing
and five days of shelf life. Therefore, faster and reliable methods
are required to detect the presence of pathogens without losing
the period of commercialisation. In the framework of the EU project
QUAFETY - Comprehensive Approach to Enhance Quality and
Safety of Ready to Eat Fresh Products different detection methods
based on ELISA approach was developed and compared with the
standard official ISO procedures.
Materials and Methods
Samples: Rocket and mixed salads were provided by Agronomia
s.r.l., (San Paolo d’Argon, BG, Italy) and EuroCatering S.A. (Greece),
while Piel De Sapo melons were provided by NoviFruits (Portugal).
Samples were collected from raw materials, processed materials
(immediately after packaging) and three days shelf-life materials.
Thirty-nine samples were analysed corresponding to 2 different
batches of melons (Portugal), 7 different batches of rocket (Italy
and Greece) and 4 different batches of mixed salad (Italy and
Greece).
ELISA-based detection of L. monocytogenes and E. coli
O157:H7: Five g of material was homogenised in 1×PBS (1 mM
KH2PO4, 154 mM NaCl, 3 mM Na2HPO4, pH 7.2). Isolation of
bacteria, ELISA detection and data analysis were performed
according to the protocol described in Cavaiuolo et al. 4. For the
detection anti-E. coli O157 (ab20976), anti-L. monocytogenes (LZA2)
(ab11439) and Polyclonal Goat anti-Mouse IgG+IgM H&L (HRP)
antibody (ab47827) were purchased from Abcam (Cambridge, UK).
ISO procedures for L. monocytogenes and E. coli O157:H7
detection: Twenty-five g of each sample was used to detect the
presence of L. monocytogenes and E. coli O157:H7 according to
ISO 11290-1:1996/Amd 1:2004 and ISO 16654:2001 5, 6, respectively.
A sample was characterised as positive for the presence of L.
monocytogenes when the identity of typical colonies isolated from
ALOA agar was verified by series of biochemical tests (Gram stain,
catalase test, oxidase reaction, hemolysis, fermentation of
rhamnose, xylose, mannitol and methyl α-D-mannopyranoside,
and a motility test). In any other case (i.e. identity not verified,
absence of typical colonies), the sample was characterised as
negative for the presence of L. monocytogenes. A sample was
characterised as positive for the presence of E. coli O157:H7 when
Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014      99
the identity of typical colonies was verified by successive
subculturing in BH agar and submittion to E. coli O157:H7 Latex
Test (Remel, Lenexa, USA). In any other case (i.e. identity not
verified, absence of typical colonies) the sample was characterised
as negative for the presence of E. coli O157:H7.
Results and Discussion
While all melon samples were negative for both bacteria (average
OD450nm 0.09, negative control (NC) OD450nm 0.06), in fresh cut
salads the ELISA method identified few suspected samples. Based
on the NC absorbance values (average OD450nm 0.101), on the
coefficient of variation (CV) among samples and assays and on
the OD450nm values of different concentrations of E. coli O157:H7
and L. monocytogenes reported in the work of Cavaiuolo et al. 4,
the thresholds were set for classifying the samples as sure
negative, suspect and sure positive. Samples with and OD450nm≤0.3
were considered sure negative and samples with OD450nm≥0.5 were
considered sure positive for E. coli O157:H7. Only three samples
were considered as suspect: Rocket raw (OD450nm 0.36), mix raw
(OD450nm 0.321) and mix three days-shelf (OD450nm 0.322). Samples
with and OD450nm≤0.5 were considered sure negative and samples
with OD450nm≥0.6 were considered sure positive for L. monocytogenes
(Table 1). Based on these thresholds, two suspected samples were
isolated: Mix salad raw (OD450nm 0.52) and mix three days shelf
(OD450nm of 0.566).
The use of classical microbiological approaches verified the
presence of E. coli O157:H7 in mixed salad raw and rocket three
days shelf-life and absence in the rest of the samples. Absence of
L. monocytogenes was verified in all samples.
If the samples classified as ‘suspects’ are considered positives,
then a 2×2 contingency table summarising the results obtained
by the ELISA can be formed and the samples can be differentiated
into true positives, true negatives, false positives and false
negatives (Table 2). Furthermore, the performance indices of the
newly developed methods used to detect L. monocytogenes and
E. coli O157:H7 can be calculated (Table 3).
 TP TN FP FN 
L. monocytogenes 2 37 2 0 
E. coli O157:H7 3 35 2 1 
Table 2. The 2 × 2 contingency table obtained from the
application of the newly ELISA method used to
detect L. monocytogenes and E. coli O157:H7.
TP: True positive. TN: True negative. FP: False positive. FN: False negative.
Conclusions
Results obtained exhibited the technical efficiency of the
developed methods. More accurately, all methods compared had
similar sensitivity, specificity, negative predictive values and
negative likelihood ratios. False positive results obtained by the
ELISA method resulted in the reduction of positive predictive
values. Regarding their efficacy and viability for routine analysis
it is mostly dependent upon available equipment and technical
expertise.
Acknowledgements
The research leading to these results has received funding from
the European Union Seventh Framework Programme (FP7/2007-
2013) under grant agreement no. 289719 (Project QUAFETY). All
authors contributed equally to this publication.
References
1Rico, D., Martín-Diana, A. B., Barat, J. M. and Barry-Ryan, C. 2007.
Extending and measuring the quality of fresh-cut fruit and vegetables:
A review. Trends Food Sci. Tech. 18(7):373-386.
2Oliveira, M., Usall, J., Vinas, I., Anguera, M., Gatius, F. and Abadias,
M. 2010. Microbiological quality of fresh lettuce from organic and
conventional production. Food Microbiol. 27(5):679-684.
3Goodburn, C. and Wallace, C. A. 2013. The microbiological efficacy of
decontamination methodologies for fresh produce. Food Control
32(2):418-427.
4Cavaiuolo, M., Paramithiotis, S., Drosinos, E. H. and Ferrante, A. 2013.
Development and optimization of an ELISA based method to detect
Listeria monocytogenes and Escherichia coli O157 in fresh vegetables.
Anal. Methods 5(18):4622-4627.
5International Organization for Standardization (ISO) 1996. Microbiology
of Food and Animal Feeding Stuff - Horizontal Method for the
Detection and Enumeration of Listeria monocytogenes. Part 1:
Detection Method. Geneva, Switzerland.
6International Organization for Standardization (ISO) 2001. Microbiology
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Detection of Escherichia coli O157. Geneva, Switzerland.
Table 1. Detection of E. coli O157:H7 and L. monocytogenes in
rocket and mix salad samples using the ELISA method.
E.coli 0157:H7 L. monocytogenes 
Sample 
OD450nm 
mean 
Stdev 
 
OD450nm 
mean 
Stdev 
 
Rocket raw 0.360 0.204 Suspect 0.449 0.181 Negative 
Rocket processed 0.152 0.08 Negative 0.203 0.138 Negative 
Rocket shelf-life 0.202 0.105 Negative 0.285 0.171 Negative 
Mix raw 0.321 0.08 Suspect 0.519 0.148 Suspect 
Mix processed 0.242 0.05 Negative 0.419 0.202 Negative 
Mix shelf-life 0.322 0.06 Suspect 0.566 0.228 Suspect 
Negative 0.101 0.017 0.095 0.030 
Melon raw 0.091 0.002 Negative 0.076 0.025 Negative 
Melon processed 0.087 0.001 Negative 0.088 0.002 Negative 
Melon shelf-life 0.094 0.001 Negative 0.092 0.001 Negative 
Negative 0.061 0.003  0.063 0.001  
 L. monocytogenes E. coli O157:H7 
Sensitivity 1 0.75 
Specificity 0.94 0.94 
Positive predictive value 0.5 0.06 
Negative predictive value 1 0.97 
Positive likelihood ratio 19.5 13.87 
Negative likelihood ratio 0 0.26 
Table 3. Performance indices of the newly ELISA method
used to detect L. monocytogenes and E. coli
O157:H7.


Validation of ELISA-based detection of L. monocytogenes and E. coli O157:H7 in fresh cut vegetables

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Validation of ELISA-based detection of L. monocytogenes and E. coli O157:H7 in fresh cut vegetables

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