This document discusses various methods for detecting foodborne pathogens. It begins by defining foodborne pathogens and explaining their impact on public health. It then describes why detecting these pathogens in food is important for food safety and quality control. The document proceeds to discuss several methods for detecting pathogens, including ELISA, DNA chips, ATP bioluminescence, flow cytometry using fluorescent labels, biosensors, PCR and its variants, and techniques for detecting specific pathogens like Salmonella, Listeria, E. coli, and Staphylococcus aureus. Advantages and disadvantages of these various detection methods are also summarized.

1. FOOD SAFETY
BASICSTEPS IN DETECTION OF FOOD BORNE PATHOGENS
GROUP _5
BHAVNA VIJAYAN
LEENA
NEHA
SURBHI RAI
SAUMYA JAIN

2. FOOD BORNE PATHOGENS
According to NCBI, “Foodborne pathogens (e.g. viruses, bacteria, parasites) are
biological agents that can cause a foodborne illness event. A foodborne disease
outbreak is defined as the occurrence of two or more cases of similar illness
resulting from the ingestion of a common food”.
Furthermore the 31 pathogens identified as causing foodborne illnesses, Salmonella,
Campylobacter, Staphylococcus aureus, Listeria monocytogenes, Clostridium
perfringens and Escherichia coli have been incriminated for the large majority of
illnesses, hospitalizations and deaths. And even among these Salmonella spp, L.
monocytogenes, E.coli and S. aureus account for causing largest number of
outbreaks, cases and deaths.

3. NEED OF DETECTING FOOD BORNE
PATHOGENS
 Microbiological examination of food is necessary from safety point of view as food may act
as a vehicle for transmission of pathogenic microorganisms.
 The microbial load present in food at different stages of its processing also needs to be
checked in order to keep a check on quality of the ingredients being used in food and also
food as a whole.
 Quality of some foods is also related to the number and type of microorganisms present in
food like probiotic food products require a high number of viable bacteria of the specific
strain so as to provide claimed health benefits.
 Also food pathogens or in broader sense food microorganisms need to be kept in check in
minimally processed, ready to eat foods, dairy products prepared from unpasteurized milk
and fruits and vegetables.
 The foodborne pathogens can enter through contaminated water or contaminated and
undercooked food. Hence, it is important to detect the presence of pathogens in food and
water before it enters the body to cause a serious outbreak

4. Steps involved in food analysis for pathogen detection

5. ELISA ( Enzyme Linked Immunosorbent Assay)
 Enzyme-Linked Immunosorbent Assay (ELISA) is an immunological technique used for detecting and measuring
specific proteins, such as antibodies, antigens, and hormones in biological samples.
 The basic steps of ELISA are:
 Immobilization of the target proteins/antigens on the surface of a microplate
 Washing unbound/excess proteins/antigens from the plate
 Adding a labeled antibody which will subsequently bind the target antigen/protein present in the plate
 Washing unbound (excess) antibodies off the plate
 Adding enzyme-specific substrates that will react with the enzyme and produce a colored product, which
can be measured colorimetrically using a microplate reader.

6. Types of ELISA DIRECT ELISA
INDIRECT ELISA
SANDWICH ELISA ( Most effective)
COMPETITIVE ELISA (when antigen are
in low conc .)

7. DNA CHIPS AND GENOMICS -:
DNA chips are a combination of semiconductor technology and molecular biology. DNA chips consist of
large arrays of oligonucleotides on a solid support. They are prepared by one of three methods :
(1) Growing oligonucleotides on the surface, base by base. This is called a GenechipTM.
(2) Linking presynthesised oligonucleotides or PCR products to a surface.
(3) Attaching such materials within a small, three-dimensional spot of gel. .
DNA chip technology also makes it possible to detect diverse individual sequences simultaneously in
complex DNA samples. Therefore, it will be possible to detect and type different bacterial species in a
single food sample
ATP bioluminescence techniques and hygiene monitoring
 The molecule adenosine triphosphate (ATP) is found in all living cells (eucaryotic and procaryotic).
Therefore the presence of ATP indicates that living cells are present. The limit of detection is around
l pg ATP, which is equivalent to approximately 1000 bacterial cells based on the assumption of 10- l5 g
ATP per cell
 Since a sample is analysed in seconds to minutes it is considerably faster than conventional colony
counts for the detection of bacteria, yeast and fungi. Additionally, food residues which act as theIr
loci of microbial growth will also be detected rapidly
 ATP bioluminescence is primarily used as a hygiene monitoring method and not for the detection of
bacteria ATP bioluminescence can be used as a means of monitoring the cleaning regime, especially
at a Critical Control Point of a Hazard Analysis

8. PROTEINDETECTION
 An alternative to ATP detection for hygiene monitoring is the detection of protein residues,
using the Biuret reaction
 The surface is sampled either by swabbing or by a dipstick, and reagents added. The
development of a green colour indicates a clean, hygienic surface, grey is ‘caution’ and
purple is ‘dirty’.
 The technique is more rapid than conventional microbiology and less expensive than ATP
bioluminescence since no capital equipment is required. It is, however, less sensitive than
ATP bioluminescence
FLOW CYTOMETRY:
Flow cytometry is based on light scattering by cells and fluorescent labels which discriminate
the microorganisms from background material such as food debris

9. • Fluorescence-labelled antibodies have been produced for the major food poisoning
organisms such as Salmonella serovars, L. monocytogenes, C.jejuni and B. cereus.
• The level of detection of bacteria is limited to approximately lo4 cfu/ml due to
interference and autofluorescence by food particles.
• Fluorescent labels include fluorescein isothiocyanate (FITC), rhodamine isothiocyanate
and phycobiliproteins such as phycoerythrin and phycocyanin. These emit light at
530nm, 615 nm, 590 nm and 630 nm, respectively.
• Viable counts are obtained using carboxyfluorescein diacetate which intracellular
enzymes will hydrolyse, releasing a fluorochrome.
• Fluorescent-labelled nucleic acid probes, designed from 16s rRNA sequences, enable a
mixed population to be identified at genus, species or even strain level .
• However, as the organism might be non-culturable it is uncertain whether the
organism was viable in the test sample and subsequently questions whether its
detection is of any significance. The method has been used for the detection of viruses
in sea water

10. BIOSENSORS
 A biosensor can be defined as a quantitative or semiquantitative analytical instrumental technique containing
a sensing element of biological origin, which is either integrated within or is in intimate contact with a
physicochemical transducer.
 Biosensors is an analytical device that consists of two main elements:-
1)Bioreceptor-enzyme, antibody, aptamer etc- Responsible for recognition
2)Transducer- Convert Biological interaction into a measurable electrical signal.
 Based on the type of transducer, biosensor maybe of different types
1) Optical biosensor
2) Electrochemical biosensor
3) Mass based biosensor

11.  Potentiometric, amperometric and conductometric types of biosensors all come under electrochemical
biosensors.
 The bioreceptor responsible for recognizing the target analyte can either be a:
1. Biological material: enzymes, antibodies, nucleic acids and cell receptors, or
2. Biologically derived material: aptamers and recombinant antibodies, or
3. Bio mimic: imprinted polymers and synthetic catalysts.

12. OPTICAL BIOSENSORS
 The most commonly used optical biosensor for the detection of foodborne pathogen is
surface plasmon resonance (SPR) biosensor due to their sensitivity.
 SPR employs reflectance spectroscopy for the pathogen detection.
 In SPR, bioreceptors are immobilized on the surface of a thin metal.
 The electromagnetic radiation of a certain wavelength interacts with the electron cloud
of the thin metal and produces a strong resonance.
 When the pathogen binds to the metal surface, this interaction alters its refractive index
which results in the change of wavelength required for electron resonance.

13. OPTICAL BIOSENSORS
 Optical biosensors are also known as “optodes” because of their resemblance with
electrodes.
 These include determining the changes in light absorption between the reactants and
products of a reaction, or measuring the light output by a luminescent process.
 Optical biosensors integrate optical technique with a biological element to identify
chemical or biological species.
 This technique is employed to monitor pesticides, vitamins, carcinogens and toxins
based on chemiluminescence and fluorescence.

14. ELECTROCHEMICAL BIOSENSORS
 Electrochemical transducer where electrochemical signals are generated during
biochemical reactions and are monitored using suitable potentiometric, amperometric or
conductometric systems of analyses.
 It is considered as a chemically modified electrode since electronic conducting,
semiconducting or ionic conducting material is coated with a biochemical film.
 Many enzyme reactions, such as those of urease and many biological membrane
receptors may be monitored by ion conductometric or impedimetric devices, using
interdigitated microelectrodes.
 As the sensitivity of the measurement of hindered by the parallel conductance of the
sample solution, usually a differential measurement is performed between a sensor with
enzyme and an identical one without enzyme. Analytes like urea, charged species and
oligonucleotides are detected using this principle.

15. MASS BASED BIOSENSOR
 Mass-based or mass-sensitive biosensors operate based on the detection of small
changes in mass.
 Mass-based biosensors involve the use of piezoelectric crystal which will vibrate at a
certain frequency when induced by an electrical signal of a certain frequency.
 The bioreceptors (e.g., antibodies) for the detection of pathogens (e.g., antigens) are
immobilized on this crystal.
 Once the target antigens bind to the antibodies immobilized on the crystal, this will
cause a measurable change in the vibrational frequency of the crystal which correlates
with the added mass on the crystal surface.
 There are two major types of mass-based biosensors which are the bulk acoustic wave
resonators (BAW) or quartz crystal microbalance (QCM) and surface acoustic wave
resonators (SAW)

16. ADVANTAGES OF USING BIOSENSORS
 Biosensors are easy to operate and they do not require sample pre-enrichment, unlike
nucleic-acid based methods and immunological methods which require sample pre-
enrichment for concentrating the pathogens before detection.
 It is a rapid means to detect foodborne pathogens and also has a high selectivity towards
targeted ions.
 Biosensors have a wide linear range of sensor response/detection limit.

17. DISADVANTAGES OF USING BIOSENSORS
 The commercialization of biosensors is slower than other rapid methods due to several
factors such as cost consideration, quality assurance, stability issues, sensitivity issues
and instrumentation design.
 There are difficulties in the methods of producing inexpensive and reliable sensors, the
storage of biosensors, the stabilization of biosensors, methods of sensor calibration and
total integration of the sensor system.
 Temperature, pH conditions tend to affect the reading one gets using biosensors.

18. Separation and concentration of target organism
Purpose – to shorten detection time and improve specificity.
(1). Immunomagnetic separation (IMS)
- uses superparamagnetic particles coated with antibodies against
the target organism.
- function through antigen-antibody specificity.
- the selective enrichment step (overnight) is replaced by thy
immunomagnetic separation (10 min).
- Commercially available IMS kits for Salmonella spp, E.coli 0157:H7,
L.monocytogens etc.
- In conventional IMS the magnetic is used outside the test-tube which
decrease the efficiency ,but in Pick Pen IMS the magnetic is present
intrasolution thus increases the throughput of the process.

19. .

20. (2). Direct Epiflurorescent technique (DEFT)
- Uses membrane filters for detection
- membranes can be made from nitrocellulose, cellulose acetate esters , nylon,
polyvinyl chloride and polyester.
- Acridine orange is used , viable cells fluoresce orange red whereas dead cells
fluoresce green.
- DEFT is a sensitive and rapid method for detecting milk and other dairy
products , complete result in 25-30 minutes and as few as 6 × 103 bacteria / ml of
raw milk or other dairy product.
(3). Hydrophobic grid membrane filters (HGMF)
- also uses membrane filters
- membrane filters traps microorganism on a membrane in a grid of 1600
compartments, due to hydrophobic effects.
- afterwards placed on a afar surface and incubated and colony count is
determined.

21. PCR (Polymerase chain reaction)
-Discovered by Kary Mullis in 1985
- in PCR there is no need to emphasis on selective media
-Principle is to amplify the genes of target pathogen and then identification
-In PCR method a heat stable DNA polymerase Taq , DNA primer and nucleotides are used
-Consists of three steps in each cycle
(a) Denaturation – 94°C for 5 min
(b) Annealing – 55°C for 30 sec
( c) Extension – 72°C for 2 min
-Cycles of these 3 steps can be repeated 30-40 times
-After completion of all cycles the DNA is stained with ethidium bromide and visualized by Agarose gel
electrophoresis with UV transillumination at 312 nm
-If the target have RNA is a DNA copy is made by using reverse transcriptase
-Advantage- Rapid, sensitive then traditional and culture based method
-Disadvantage-
 Includes cell lysis and nucleic acid extraction, cross contamination and failed due to presence of
inhibitory substance or competing DNA from the non target cells.
 PCR methods (conventional) are not able to differentiate between the live and dead cells

22. Different types of PCR used
a. Simple PCR
b. multiplex PCR
c. Oligonucleotide DNA microassay
both b & c can detect five or more pathogens
simultaneously
d. Real time PCR – a PCR based technique that couples
amplification of a target DNA sequence with
quantification of the concentration of that DNA species
in the reaction
e. DIANA – Detection of Immobilised Amplified Nucleic acids
- a variation of PCR uses 2 primer out of which only
inner one is labelled.
f. LAMP- loop mediated isothermal amplification
-employs 4-6 designed primers and a
strand-displacing Bst DNA polymerase to amplify up
to 109 target DNA copies under isothermal condition
(60-65°C)
g. NASBA – nucleic acid sequence based amplification

23. https://www.youtube.com/watch?v=iQsu3Kz9NYo&list=LL&index=2 PCR
https://www.youtube.com/watch?v=vtxb6Tr8Y3s&list=LL&index=1 AGE
Detection of Staphylococcus.aureus
-It is a facultative aerobes, gram positive coccus.
- It is highly vulnerable to destruction by heat treatment and nearly all sanitizing agents, thus its
presence or its enterotoxin in processed foods or on food processing equipment is generally an
indication of poor sanitation
-For its detection enrichment is not used.
-Viable cells test is done for sample before heat treatment and test is done for the enterotoxin and
heat stable thermonuclease for heat-treated samples
- (A). Agar plates
Baired Parker agar
- medium contains sodium pyruvate (to aid resuscitation of injured cells),selectivity is due to
tellurite, lithium chloride and glycine (growth stimulant ,an essential part of cell wall)
- It forms black colonies due to tellurite reduction and clearance of egg yolk due to lipase
activity
Mannitol salt agar
- selective agent is salt (7.5%) and mannitol fermentation is indicated by ph indicator red

24. (B). Coagulase test
- S.aureus is known to produce coagulase, which can clot plasma into gel in tube to agglutinate cocci
on slide
-It produce two types of coagulase free (extracellular) and bound (cell wall associated)
-Free coagulase is detected in tube and bound in slide coagulase test
-Free coagulase is heat labile while bound coagulase is heat stable
-In test the test sample is treated with a drop of plasma and mixed well and, If within 5-10 sec there is
agglutination or clumping of cooci the sample is taken as positive.

25. (C) DNAse test
-Colonies producing DNAse hydrolyse the deoxyribonucleic acid (DNA) content of the medium located in
their vicinity and is indication of pathogenicity.
-DNA and toluidine blue or methyl green are included in the agar medium.
-The dyes form coloured complexes with the DNA and hence there are zones of decolourisation DNA
degrading S.aureus colonies.
(D) PCR
(E) ELISA – detection limit of 0.5 µg toxin per 100g food and give result in 7 hrs
(F) Reverse latex agglutinations – limit of sensitivity is about 0.5 ng of enterotoxin per
gram of food

26. SALMONELLA :-
The salmonellae are gram-negative, non-spore-forming rod-shaped bacteria
belonging to the family Enterobacteriaceae. However, Salmonella is not included in the group of
organisms referred to as coliforms.
Salmonella is one of the principal cause of food – borne gastroenteritis and is also an important
pathogen of livestock
Salmonellosis is a zoonotic infection (can be transmitted to humans from animals)
Many microbiologists now use a classification that recognises only two species of Salmonella. These
are S. enterica (which includes six subspecies) and S. bongori. The subspecies most important in
food-borne disease is S. enterica subspecies enterica
Food animals can become infected with Salmonella from feed and from the environment, and many
foods of animal origin such as meat, poultry, eggs and raw milk can be contaminated with the
pathogen. . For example, in 2005 an EU-wide study found that about one in five large-scale
commercial egg producing facilities had hens infected with Salmonella, with the lowest levels of
infection being found in Sweden and Luxembourg, and the highest levels in Portugal, Poland and the
Czech Republic.
• Fresh produce may also become contaminated with Salmonella from animals and environmental
sources.

27. • Cooked ready-to-eat foods can become contaminated as the result of cross contamination
from raw food
• . Although contamination can occur as the result of direct contact, it can also occur via
food preparation surfaces or equipment used for both raw and cooked foods.
• . A wide variety of processed foods have been found to be contaminated with Salmonella,
including chocolate, breakfast cereal, flavoured potato crisps and similar snack products,
peanut butter, fermented meats, cheeses, milk powder and ice cream.
EFFECT ON HEALTH-:
• The more usual food-borne form of the illness is caused by non-typhoid salmonellae,
which invade the cells lining the small intestine. These organisms cause gastroenteritis
lasting between 1–7 days, with symptoms that include diarrhoea, abdominal pains,
nausea, vomiting, and chills, leading to dehydration and headaches.
• Individuals recovering from salmonellosis can continue to shed Salmonella in their stools
for some time.
• Some Salmonella serotypes have a limited host spectrum such as S. Typhi and S. Paratyphi
in humans (causing typhoid fever), S. Dublin in cattle, and S. Choleraesuis in pigs.

28. Escherichia coli identification
 Escherichia coli can act as an indicator for the presence of other pathogenic bacteria,
and it is detected easily in foods such as pork, beef, and chicken. Thus, E. coli detection
in foods is one of the most useful hygienic criteria.
 Polymerase chain reaction (PCR), using primers against the uidA gene that encodes
beta-D-glucuronidase can be used to identify E. coli accurately. PCR detection method
has been used to identify a colony on an agar plate, which was formed by plating at
least 24-h enriched broth. In addition, there is an issue of specificity, since uidA gene is
also present in Shigella

29. PROCEDURE
 E. coli samples are extracted from different sources and cultured in tryptic soy broth.
 1 ml aliquots of the enriched samples were plated onto E. coli/coliform petrifilm to quantify E. coli. The
plates were incubated at 37°C for 24 hours, and colonies were manually counted.
 One-milliliter aliquots of inoculated and enriched samples were centrifuged and supernatants were
discarded. Cell pellets were resuspended in 30 µL distilled water and boiled. The suspensions were
centrifuged for 3 min. The supernatants were then used for PCR analysis.
 Primers targeting the uidA and Shigella identification gene were used to differentiate E. coli from Shigella.

30.  PCR conditions were as follows: 94°C for 2 min (initial denaturation), 94°C for 20 s
(denaturation), 72°C for 20 s (extension), and 72°C for 2 min (final extension).
 Annealing was performed at 53°C for uidA or at 62°C for the Shigella identification
gene for 10 s, and 35 cycles were performed. PCR analysis was performed using Fast
mix French PCR and PCR products were run on an agarose gel (1.5%) with
electrophoresis for 20 min.
 Target bands were visualized under UV light.
 Thus the combination of enrichment and PCR detection method is useful to detect E.
coli via applying PCR with uidA primers to samples directly after 5-h enrichment for
fresh meats (pork and beef) and 3-h enrichment for fresh-cut lettuce.

31. Detection of Shigella
 Causes shigellosis
 The culture-based techniques were used as the gold standard for the
detection of Shigella spp. in various samples, but the conventional procedures
required multiple subculture steps, biochemical and serological confirmation,
which took about 7 days, and were time-consuming and laborious .
 In recent years, molecular technologies, such as PCR and real-time PCR assays,
have been successfully applied to detect Shigella sp . (But these techniques
can't be used in poor regions which don’t have specialized labs , trained people
and expensive reagents . So it is difficult to develop a rapid detection method
for this species .

32. Traditional method for Shigella method
Taking sample Pre enrichment
Selective
enrichment
Selective plating
Biochemical
characterization
Serological
confirmation

33. Shigella detection
by PCR