18-08-2012, 05:08 PM
Development of a Multiplex PCR Method for the Detection of
Six Common Foodborne Pathogens
Detection of six foodborne pathogens in food using MPCR.pdf (Size: 353.51 KB / Downloads: 188)
ABSTRACT
This study developed a multiplex PCR method for the screening and detection of six common foodborne pathogens in Macao.
The m-PCR procedure, which uses six pairs of primers, produced specific amplicons of the expected sizes from mixed populations
of reference bacterial strains in food samples and from pure cultures. The verocytotoxin (stx) gene of Escherichia coli O157: H7, the
hemolysin (hly) gene of Listeria monocytogenes, the invasion (invA) gene of Salmonella spp., the cholera toxin (ctx) gene of Vibrio
cholerae, the thermolabile hemolysin (tlh) gene of V. parahaemolyticus, and the thermostable nuclease (nuc) gene of Staphylococcus
aureus were used as target genes for m-PCR detection. The detection limit of the assay for the bacterial targets was 1-100 cfu per
mL. The m-PCR analysis was designed for three main food clusters; meat and meat products testing for Salmonella spp., L. monocytogenes,
and E. coli O157: H7, seafood and seafood products testing for V. cholerae and V. parahaemolyticus and ready-to-eat foods
testing for S. aureus. Overall, results of the present study indicate that the m-PCR is a potential technique for the rapid detection of
foodborne bacteria for routine monitoring and risk assessment of food.
Key words: foodborne, bacterial pathogens, multiplex PCR
INTRODUCTION
Surveillance of foodborne diseases is of an increasingly
high priority in the public health agenda worldwide.
Bacterial contamination of food represents one of the major
public health problems. Staphylococcus aureus, Salmonella
spp., Escherichia coli O157: H7, Listeria monocytogenes,
Vibrio cholerae, and V. parahaemolyticus are the main
pathogens involved in food poisoning and are routinely
monitored in Macao(1). Some of these foodborne pathogens
can cause life-threatening diseases to humans and animals.
Whilst all are well recognized, some are considered emerging
because they have recently become more common.
Salmonella spp., S. aureus, E. coli O157: H7 and L.
monocytogenes are the predominant bacteria species that
cause public health problems worldwide. In addition, V.
cholerae is a common bacterium in raw or under processed
seafood which can cause very severe diseases, and has been
endemic in Asia and Africa for years. V. parahaemolyticus
is also a frequent cause of food poisoning in seafood, thus
V. cholerae and V. parahaemolyticus are significant pathogens
that require testing in seafood or related products.
The Food & Drug Administration’s Bacteriological
Analytical Manual (BAM)/AOAC international standard
culture methods(2) are used in the government laboratories
of Macao as the golden standard. A number of immunological
protocols have also been developed for the detection
of bacterial pathogens. The VIDAS enzyme-linked
immunofluorescent assay (ELFA) is the official AOAC
method for the screening of Salmonella spp., E. coli O157:
H7 and L. monocytogenes in routine samples, but the tests
are mono-specific hence separate tests for each bacterium.
Since commercial kits are unavailable for V. cholerae
and V. parahaemolyticus, traditional culture-based
methods are required, which are time-consuming, tedious,
low throughput and invariably mono-specific (detecting
only one type of pathogen at a time). It takes at least four
days to identify the species and more than four media for
enumeration and selective culture for each pathogen.
The use of molecular methods has provided highly
sensitive detection methods for specific pathogens in
environmental samples. Numerous studies have been
published on m-PCR detection of foodborne pathogens
including pathogenic E. coli(3), E. coli O157: H7(4), Salmonella
and Shigella(5), V. parahaemolyticus(6), E. coli, S.
Typhimurium and Vibrio spp.(7), S. aureus and Yersinia
enteroliticas(8), Listeria spp.(9) Campylobacter jejuni and
Arcobacter butzleri(10).
Most of these m-PCR studies have been developed
for foodborne pathogens commonly encountered in the
reported areas or artificially inoculated(11). Hence, it is
necessary to develop m-PCR protocols which fit the local
* Author for correspondence. Tel: +853-66837838; 853-3998626
Fax: +853-28753159; E-mail: iflei[at]ipm.edu.mo
Journal of Food and Drug Analysis, Vol. 16, No. 4, 2008
38
situation. In this study an m-PCR assay was designed
to detect the six most common foodborne pathogens in
Macao. The m-PCR test detects Salmonella spp., L monocytogenes,
and E. coli O157: H7 in meat and meat products,
V. cholerae and V. parahaemolyticus in seafood and
seafood products, and S. aureus in ready-to-eat foods.
MATERIALS AND METHODS
I. Bacterial Strains and Cultural Methods
The bacterial strains examined in this study are
listed in Table 1. Sources were obtained from the American
Type Culture Collection, World Health Organization,
local hospitals and municipal laboratory. All bacterial
strains were cultured in tryptic soy broth yeast extract
broth (TSBYE) at 35°C(12). The TSBYE contained 30 g
of tryptic soy broth powder (Difco Laboratories, Detroit,
MI, USA), 6 g of yeast extract, and 1 litre of water.
II. Food Sample Preparation
Food samples were either purchased from local food
stores, collected by Health Bureau, or obtained from local
food microbiology laboratory and homogenized follow-