1
Revolution of Molecular Techniques in detection of Foodborne Pathogens
Marwa M. Radwan1 and Zeinab A. Sayed-Ahmed2
1
Department of Food Hygiene, Alexandria Food inspection Laboratory, Animal Health Research
Institute, Egypt.
2
Food Hygiene Research Unit, Alexandria Provincial Lab, Animal Health Research Institute,
Egypt.
Abstract
Introduction: Food borne pathogens like bacteria, viruses, fungi, and some
parasites are widely spread and become a major public health issue worldwide.
These pathogens have significance impact on human health. Many human diseases
originated from consumption of contaminated food. Traditional methods which
include (pre-enrichment, selective enrichment, selective plating, biochemical
screening and serological confirmation) are accurate and considered to be gold
standard methods, unfortunately, they are time-consuming, laborious, not highly
specific, low sensitivity. So, there is an urgent need for rapid, accurate, highly
specific, highly sensitive techniques like molecular techniques. These molecular
techniques have been developed for foodborne pathogens including nucleic-based
methods, immunological methods and biosensor-based methods.
Aim of work: Review some of the most novel methods of nucleic-based methods
and bio-sensor based methods that used for detection of food borne pathogens.
Key words: Food borne pathogens_ molecular techniques_ nucleic-based
methods_ bio-sensor based methods.
Introduction:
Foodborne pathogens which involve all types of microorganism that make
contamination of food or water and cause illnesses to human ,microorganism may
include (Bacteria, fungi, viruses) as well as parasites ( Dwivedi and Jaykus, 2011).
Nowadays, diseases that are caused by foodborne pathogens become one of the
most important public health problems, causing a significant rate of morbidity,
mortality, hospitalization cases, outbreak cases, infection cases and in sever cases
lead to death, additionally, diarrhea that is a main reason of malnutrition in infants
and children (WHO,2007).
In spite of advances in pathogenic detection , foodborne illness still more common
problem in a lot of countries, in addition to the Centers for Diseases Control and
Prevention reported cases of foodborne illnesses caused by unknown pathogenic
agents which can rise to high rate of mortality ( CDC,2016).
2
Some of the most common pathogens that associated with foodborne illness are
Noroviruse (Koo et al, 2010), Salmonella spp (Scallan et al, 2011),Campylobacter
spp, Escherichia coli O157:H7, Clostridium perfringens , Toxoplasma gondii,
Staphylococcus aureus, and Listeria ivanovii (Alvarez-Ordónez et al .,2015).
Conventional methods for detection of pathogens from food which based on
culturing the organisms on agar plates and then identified by biochemical
confirmation, taking a long of time may extend to more than 7 days so it was clear
that the need to methods saving time and more sensitive and specific, thus Novel
molecular detection methods are being developed as a rapid, sensitive, and
selective one , the rapid molecular methods can be classified into, nucleic-based
method, immunological methods and biosensor-based method.
The aim of this paper to review this rapid methods of detection and identification
focusing on nucleic and biosensor-based methods with described the advantages
,disadvantages and limitation of each one.
Nucleic acid-based methods
Nucleic acid-based methods are highly specific, depend on detection of specific
nucleotides sequence in target nucleic acid by hybridizing it to complementary
synthetic oligonucleotide sequence (probes or primers), furthermore, these
methods can detect toxin related genes in many toxin-producing bacterial
pathogens (Zhao et al., 2014).
These methods are advantageous in simplicity, time-saving and stable results
(Moreira et al., 2007).There are many nucleic acid-based methods but only probe
hybridization and nucleic acid amplification techniques have been developed
commercially for detecting foodborne pathogens (López-Campos et al., 2012).
Nucleic acid hybridization
Nucleic acid hybridization is a technique used for identification of nucleic acid
without amplification. It based on high specificity of base pairing between
homologous single stranded DNA (Eissa., 2012). This technique requires labeled
nucleic acid probe to detect the target DNA or RNA within mixture of unlabeled
nucleic acid molecules, the stability of target-probe hybrid depend on the extend of
base pairing occur (Strachan., 1999).
Typically, the hybridization process operates by denaturation of target DNA either
by high temperature (above 95 °C) or by high pH (above 12), followed by addition
of labeled gene probe (if the probe has oligonucleotide sequence complementary to
target DNA, the probe will bind specifically to target DNA forming probe-target
hybrid), washing step takes place to eliminate the unhybridized probe (Laizrd et
3
al., 1991). The hybrid can be visualized by the labeled probe. The intensity of the
spot is proportional to concentration of hybridized probe and consequently is
proportional to the concentration of target DNA in the sample. The intensity can be
compared visually with the intensity of spot in standard curve giving semiquantitative results (visual quantification), or measure the intensity by instrument
(e.g. densitometer) resulting in quantitative value (Olsen et al., 1995)
The major disadvantage of hybridization techniques is lack of sensitivity, which
limit their use to population of cells or genes that occur with relatively high
numbers in the sample. For this reason hybridization techniques used mainly for
conformation of culture rather than direct detection and identification (Eissa,
2012).
Amongst the hybridization techniques, a particular focus should be given to
fluorescent in situ hybridization (FISH).
FISH requires fluorescent labeled ribosomal RNA (rRNA) probe and fluorescent
microscope in detection of bacteria directly in food or after enrichment culture
(Amann et al., 1990a; Amann et al., 1990b).
FISH steps include preparation of sample by fixation and per-meabilisation,
binding of the probe, washing to remove unbound probes, finally detection of
hybridized probe by flow cytometer or microscopically (Amann et al., 2001).
Nucleic acid amplification
Nucleic acid amplification techniques have many applications and become a
fundamental tool in rapid detection of foodborne pathogens. These techniques
based on amplifying target nucleic acid sequence in vitro with high sensitivity of
detection down to one copy of nucleic acid in reaction, also may quantify it in
many cases (Liu, 2010).
The amplification assays include PCR-based amplification and non PCR-based
amplification (Wang and Salazar, 2016).
PCR-based amplification
PCR-based assays become indispensible tools in rapid detection, identification and
differentiation of foodborne pathogens (Adzitey et al., 2013).
Polymerase chain reaction (PCR) was developed in 1986 as a method for in vitro
amplification of DNA (Mullis, 1990). This reaction operates by repeating thermal
4
cycles, the products of each cycle serve as template for next cycle, lead to
duplication of initial number of DNA molecules with each cycle (Hill, 1996).
There are many variants of PCR as simple PCR, multiplex PCR, real time PCR,
nested PCR, reverse transcripted PCR.
Simple PCR
One of the most powerful analytical techniques ever been developed is PCR which
creates several million fold of target DNA molecule from minute amount of
double-stranded DNA (dsDNA) within several hours. Its most prominent
application is detection of low numbers of foodborne pathogens and toxinproducing bacteria in various food types as well as conformation of identified
pathogens from food (Levin, 2010).
PCR reaction needs a thermostable DNA polymerase, deoxyribonucleoside
triphosphates (dNTPs), primers (two single strand synthetic oligonucleotide
complementary to target DNA sequence), magnesium chloride and template or
target DNA (Liu., 2010). This reaction performs in three sequential steps (Olsen et
al., 1995), begins with denaturation of template DNA (double stranded DNA) into
two single strands, followed by annealing of primer to target DNA sequence,
finally extension of primer in presence of DNA polymerase and nucleotides to
form complementary strand (Zhao et al., 2014). A typical amplification process
need 20 – 40 cycles (Shi et al., 2010). The PCR products can be seen as a band on
gel-electrophoresis stained with ethidium-bromide (Priyanka et al., 2016)
PCR is rapid, sensitive, specific and operates with small quantities of sample
comparable to culturing methods (Hill, 1996).
Multiplex PCR (mPCR)
Multiplex PCR is a modification of simple PCR, it was established in 1988
(Chamberlain et al., 1988). mPCR has been used with a wide range in detection of
foodborne pathogens (wang and Salazar, 2016). The assay includes simultaneous
amplification of multiple target DNA sequences by multiple sets of primers which
are included in a single reaction tube ( Bej et al., 1991).
The major factor in the development of mPCR is the designing of the primers, that
they must have a very close annealing temperatures (Shi et al., 2010), as well as
their concentration, must be adjusted in order to prevent non-specific interaction
between multiple primers (primer dimer) and consequently provide a reliable PCR
5
products (Zhao et al., 2014). Moreover, the design should implement techniques to
differentiate between amplicons after thermal cycling. The techniques may include
designing of target sequence with different sizes or melting temperature that
distinguish by gel-electrophoresis or non-specific dyes which bind to any double
strand DNA, respectively, or by real-time PCR probes with variable excitation and
emission wave length (Liu, 2010).
Other factors are very important in implementation of mPCR like buffer
concentration, amount of template DNA, adjustment of cycles temperature,
thermostable DNA polymerase (e.g. Taq polymerase) and the balance between
magnesium chloride and nucleotides concentration ( Khoo et al., 2009).
The advantages of mPCR are cost saving (Gilbert et al., 2003); also reduce both
time and efforts (Markoulatos et al., 2002).
Real-time PCR (Rti- PCR)
Rti- PCR, also called quantitative PCR (qPCR), is considered to be a method of
choice for simultaneous detection and quantification of foodborne pathogens
(Priyanka et al., 2016), as it provide a continuous monitoring of PCR products
throughout the amplification process, which eliminates the post-PCR detection of
PCR products, and consequently reduce both detection time (compare to simple
PCR) and risk of contamination from laboratory environment (Klein and Juneja.,
1997).
Since Rti- PCR can quantify microorganism in food, it could replace plate methods
to get accurate results about bacterial load of food within few hours (Gomez et al.,
2010).
Rti- PCR based mainly on the release of an ultraviolet (UV)-induced fluorescent
signal which is proportional related the quantity of synthetized DNA (Levin,
2010). Various fluorescent systems have been developed for this purpose as SYBR
Green, TaqMan Probes, Fluorescent Resonance Energy Transfer (FRET),
Molecular Beacons and Unique Fluorogenic Primers (Levin, 2010).
Rti-PCR provide may advantages as not influenced by non-specific amplification,
amplification can be monitored at real-time, no post-PCR processing of products
(gel electrophoresis), rapid cycling, confirmation of specific amplification by
melting curve, specific, sensitive, and reproducible, but unfortunately it is high in
cost and need highly skillful persons (Park et al., 2014).
6
Non PCR-based amplification techniques:
Although PCR is the most predominant diagnostic tool in detection, identification
and differentiation of foodborne pathogens (Adziety et al., 2013), PCR have
drawbacks as unable to distinguish between live and dead cells (Wang et al.,
2001), the need for thermo-cycling that may limits their uses (Zhao et al., 2014).
Furthermore, PCR is greatly affected by certain food components like fat, lipid,
salts, enrichment media and extraction solution of DNA these can probably
interfere with PCR reaction (Wilson, 1997).
A novel isothermal amplification has been developed in last 20 years that amplify
the nucleic acid without thermal cycling, also, more tolerable to some inhibitory
substances, these may affect the amplification, than PCR. These non-PCR based
techniques are based mainly on new finding in RNA/DNA synthesis and some
associated protein and how to apply them in nucleic acid amplification in vitro
(Gill and Ghaemi, 2008).
Isothermal amplification techniques include Nucleic acid based amplification
(NASBA), loop mediated isothermal amplification (LAMP),
rolling circle amplification (RCA), strand displacement amplification (SDA).
Nucleic acid based amplification (NASBA):
NASBA has been developed by Compton in 1991 (Compton, 1991). It is one of
nucleic acid amplification techniques that amplify RNA and DNA under
isothermal condition, as it eliminate heat denaturation of the product during
amplification by using a set transcription and reverse transcription reactions (Essia,
2012).
The reaction includes three enzymes T7 RNA polymerase, RNase H and AMV
reverse transcriptase (avian myeloblastosis virus). In addition to two primers, the
first one is about 45 base pair in length and its 5` end contains a promoter sequence
that detected by T7 RNA polymerase, the second primer derived from opposite
side (5` end) of the target sequence (Compton, 1991).
The reaction takes place around 41°C Law et al.( 2015), and it includes annealing
of primer 1 to target RNA, followed by extension of the primer by AMV reverse
transcriptase that forms complementary DNA (cDNA) to RNA (cDNA-RNA
duplex). RNase H degenerates the RNA leaving a single stranded DNA to which
primer 2 anneals and extends by AMV reverse transcriptase creating double
stranded DNA molecule, rendering the promoter region double strand (Compton,
1991).
7
T7 RNA polymerase recognizes the promoter and transcripts copies of RNA from
the newly transcripted active promoter yielding as many as 100 copies of RNA
serve as templates for reverse transcriptase (Shi et al., 2010).
The end products of NASBA can be visualized by gel-electrophoresis stained with
ethidium bromide (Zhao et al., 2014), fluorescent probes (real-time NASBA) Abd
el-Galil et al. (2005) and colorimetric assay (NASBA- ELISA) (Gill et al., 2006).
NASBA owing many advantages as amplification of single- stranded RNA
directly, highly time-efficient as each transcription cycle generating 10-100 copies
of RNA comparable to PCR in which the number of target molecules doubling
with each cycle (Compton, 1991), amplification without thermo-cycling (Law et
al., 2015), contaminated DNA is not problem as there is no denaturation (Eissa.,
2012), sensitive and specific (Nadal, 2007). Moreover, real-time NASBA can
differentiate between viable and non-viable cells (Dwivedi and Jaykus., 2011).
Loop mediated isothermal amplification
LAMP is a novel isothermal amplification technique established by Notomi and
others in 2000, this novel assay able to amplify DNA under isothermal condition
with high specificity, efficiency and rapidity (Notomi et al., 2000).
LAMP based mainly on auto-cycling strand displacement DNA synthesis that
carried out by Bst DNA polymerase large fragment with high strand displacement
activity and four specially designed primers (2 inner and 2 outer) which recognize
six distinct regions in target DNA at 65 °C (Notomi et al., 2000; Zhao et al., 2014).
LAMP can produce, in less than one hour, as many as 109 copies of target DNA
from a few copies (Notomi et al., 2000).
The final LAMP products are mixture of stem-loop DNAs with multiple stem
length and cauliflower-like structure with multiple loops (Zhao et al., 2014). The
LAMP amplicons can be detected by gel-electrophoresis followed by staining with
SYBER Green 1 dye (Notomi et al., 2000), real-time turbiditmetry (Mori et al.,
2004), or by naked eye by using SYBER Green 1 dye which change color of the
solution into green color in presence of amplicons, whether not it remains orange
(Zhao et al., 2014).
Otherwise, the detection of LAMP amplicons can depend on pyrophosphate ions,
that produced in large quantities during synthesis of large amount of DNA within
short time and yielding white precipitate of magnesium pyrophosphate.
Consequently presence of white precipitate indicate amplification of DNA and
production of DNA amplicons (Mori et al., 2001) .
8
LAMP is more sensitive and specific, comparable to PCR based assays, in
detection field of foodborne pathogens, In addition to absence of both false
positive and false negative (Wang et al., 2012).
There are many variants of LAMP have been developed for detection of foodborne
pathogens as reverse transcriptase LAMP assay (Chen et al., 2008), multiplex
LAMP assay( Iseki et al., 2007), real-time reverse transcriptase LAMP assay( Liu
et al., 2009) and in situ LAMP assay (Ye et al., 2011).
Moreover, LAMP provide an efficiently rapid assay as produce large number of
amplicons (103 fold or higher) than that produced by simple PCR within less than
1 hours ( Law et al., 2015).
Biosensor-Based Methods
Biosensor is defined as an analytical device that consist of two main elements: a
bio-receptor and a transducer , the bio-receptor is responsible for recognizing the
target analyte, while the transducer is responsible for conversion of the biological
reaction into a measurable electrical signal which can be, optical ,electrochemical,
mass-based, thermometric, micro mechanical or magnetic (Zhao et al., 2014).
The target analyte can be biological material As( enzyme, antibodies, nucleic acids
and cell receptor, or Biologically derived material as aptamers and recombinant
antibodies ,or biomimic imprinted polymers and synthetic catalysts (Law et al.,
2015).
The biggest advantage of using biosensors in foodborne pathogens detection that
fast or real- time detection , portability, and multi –pathogen detection; moreover
the usage of biosensor do not require sample pre- enrichment , and that make it
unique about nuclic –acid based methods or immunological based methods.
The recent biosensors that commonly uses for detection of food borne pathogens
are optical, electrochemical and mass-based biosensors (Zhang, 2013; Zhao et al.,
2014).
Application of different types of biosensors in detection of foodborne pathogens
can be summarized in the following table
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Table: Application of biosensor- based methods for detection of foodborne pathogens in
food matrix. ( Law et al., 2015)
Detection
methods
Optical biosensor
Electrochemical
biosensors
Analyte
Detection limit
Assay time
References
Salmonella cholera suis
serotype typhimurium,
Listeria monocytogenes,
Campylobacter jejuni
and Escherichia coli
O157:H7
4.4 × 104 CFU/mL for
Salmonella cholera
suis serotype
typhimurium,3.5x 10 3
CFU/mL for Listeria
monocytogenes,
1.1x105 CFU/mL for
Campylobacter jejuni
and 1.4x104 CFU/mL
for Escherichia coli
O157:H7inPBS
103 CFU/mL
3 × 103 CFU/mL
Not stated
Taylor et al., 2006
45 min
Not stated
Wei et al., 2007
Wang et al., 2013
30 min
Chemburu et al.,
2005
15min without
enrichment; 6h
after
enrichment
Varshney et al.,
2005
Listeria monocytogenes
50 cells/mL for
Escherichia coli, 10
cells/mL for Listeria
monocytogenes and 50
cells/mL for
Campylobacter jejun
1.6 × 101–7.23 × 107
cells/mL without
enrichment and
8.0 × 100–8.0 × 101
cells/mL with
enrichement
103 CFU/mL
3h
Salmonella typhimurium
Escherichia coli
O157:H7
105–106 cells/mL
23 CFU/mL in PBS and
53 CFU/mL in milk
Kanayeva et al.,
2012
Su and Li, 2005
Shen et al., 2011
Campylobacter jejuni
Escherichia coli
O157:H7
Escherichia coli, Listeria
monocytogenes and
Campylobacter jejuni
Escherichia coli
O157:H7
Mass- based
biosensors
Not stated
4h
10
Optical Biosensors
Optical biosensors are one of the greatest conventional analytical techniques that
have, high sensitivity and specification in addition to their small size. Optical
biosensor is a compact analytical device that consist of a biological sensing
element connected to an optical transducer system (Dongyou,2010).
Many types of the optical biosensors have been developed , in the last decade for
detection of pathogens, toxins, and most contaminants of food (Velusamy et al.,
2010)
The detection technique of this biosensor depend on enzyme system, which
catalyze in conversion of analytes into products that can be reduced or oxidezed at
a working electrode and kept at specific potential .
The technology of optical biosensor classified into several subclasses based on
absorption, reflection, refraction, Raman, infrared, chemiluminescence, dispersion,
fluorescence, phosphorescene (Zhao et al., 2014 ).
The best advantage of this technique is the real- time binding detection , in addition
to the cost-effectiveness as the optical transducer has low-cost and can use biodegradable electrodes.
One of the most common methods that use the technique of optical bio sensor
using reflectance spectroscopy for detection of foodborne pathogens is surface
plasmon resonance (SPR).
the SPR technique for biosensing allows real-time monitoring of chemical and biochemical interactions occurring at the interface between a thin metal film and a
dielectric or transparent material such as the liquid analyte ( Palmiro et al ., 2014).
Receptors or antibodies immobilized on the surface of the thin metal film,
deposited on the reflecting surface of an optically transparent waveguide that used
to capture the different target pathogens . the interaction between electromagnetic
radiation of specific wavelength and the electron cloud of the thin metal create a
strong resonance. when the pathogen connects to the metal surface this interaction
change its refractive index (RI) which lead to alteration of wavelength that
required for electron resonance ( Law et al., 2015).
Availability of commercial optical biosensors using SPR technique such as
BIACORE 3000 and SPREETA biosensor helping in spreading of this technique.
SPREETA biosensor used for detection of E.Coli O157:H7 with detection limit
around 102–103 CFU/mL, Salmonella Enteritidis and Salmonella Typhimurium
11
(Lan et al., 2008). Whereas, BIACORE 3000 Biosensors used for detection of
Listeria monocytogenes with detection limit 1 × 105 cells/mL, Salmonella group
B,D, and E , E.Coli O157:H7 and Salmonella Enteritidis (Wang et al., 2011).
The main obstacles of SPR technique are its complexity (specialized staff is
required) , and the large size of most needed instruments .
Electrochemical Biosensors
Electrochemical –based methods or what is called transduction-based system
which is used for detection and quantifying foodborne pathogens .
This method depend on electrochemical impedance spectroscopy which is used as
transduction technique. the main concept of impedance biosensors that ,when the
bacterial cells are connected to the electrodes, this connection lead to changes in
the electrical properties of bacterial cells that can be measured by this biosensor(
Yang and Bashir.,2008).
Electrochemical biosensors are classified rely on the observed parameters as
current, potential, impedance, and conductance into amperometric, potentiometric,
impedimetric, and conductometric response, respectively (Velusamy et al ., 2010).
Each type of this biosensors are used successfully for detection of foodborne
pathogens, as amperometric biosensor was used for detection of Staphylococcus
aureus at detection limit 1 CFU/mL for only 2 hr, potentiometric biosensors was
used for detection of Escherichia Coli in vegetable food at detection limit 10
cells/ml , impediometric one was used for detection and quantification of
Escherichia Coli at detection limit 101- 107 CFU/mL and conductometric biosensor
was used for detection of Bacillus cereus with detection limit around 35-88
CFU/mL ( de Ávila et al., 2012)
So the main advantage of the electrochemical biosensors that can handle large
numbers of samples and automated , however, its low specificity and its need to
many washing steps may make limitation of its usage.
Mass –based Biosensor
Mass- based or mass- sensitive biosensors that include Piezoelectric biosensors,
this technique is depend on sensitive detection of minute change in mass. In
Piezoelectric biosensors as the electrical signal of a certain frequency induce the
piezoelectric crystal vibration at a certain frequency, the bio-receptor as
(antibodies) for detection of contaminants as (antigens) are immobilized on this
crystal causing a measurable change on the frequency of vibration of the crystals
that correlate with the deposited mass on the crystal surface, as there are linear
12
relationship between the deposited mass and its frequency response ( law et
al.,2015).
There are two types of Piezoelectric biosensors; the bulk acoustic wave resonance
(BAW) or quartz crystal microbalance (QCM) and surface acoustic wave
resonators (SAW) (Zhang.,2013).
(SAW) was used for detection of toxigenic E.coli O 157:H7 Berkenpas et al.(
2006), while (QCM) was reported for detection of Listeria monocytogens at
detection limit 1×107 cells/mL, and E.coli O 157:H7 at detection limit 102 CFU/mL
for analysis time less than 1.5 hr (Liu et al., 2007). Morever, salmonella
Enteritidis was detected at detection limit 1×105cells /mL and E.coli at detection
limit 106-109 CFU/mL (Si et al., 2001; Pohanka et al., 2007).
So the main advantages of the mass- based biosensor is real- time detection and
easy to operate but still the main obstacle of its low specificity , low sensitivity,
and regeneration of crystal surface may be problematic and this one reasons make
the usage of this type of biosensors for foodborne pathogen detection lesser than
electrochemical and optical biosensors ( Velusamy et al., 2010).
Conclusion
Traditional method for foodborne pathogens detection which depend on culturing
methods and biochemical confirmation ,they are time- consuming and laborious ,so
the need of rapid detection methods to overcome limitation of conventional
methods become very critical to prevent outbreak of foodborne disease and spread
of foodborne pathogens .thus, Rapid molecular detection methods are more
specific, more sensitive, more time – efficient, more labor-saving, more accurate
and effective but need trained personnel and specialized instruments.
Nucleic- acid based method and biosensor-based method, the both of them are
widely used for detection of foodborne pathogens, and combination of several
molecular method is also possible for more accurate detection of foodborne
pathogens . Rapid molecular methods give us great potential chance for control and
limitation of spread of foodborne pathogens and protect human health.
13
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