STKM2112 Food Microbiology

Molecular Analysis

Dr. Mohamed Yusuf Mohamed Nazir

Senior Lecturer
Department of Food Sciences,
Faculty of Sciences and Technology,
Universiti Kebangsaan Malaysia
 Introduction

24-48 hours Why?

Investigation
 Microbial Detection

Microbiology analysis Molecular Detection

Mapping of rapid detection technologies for foodborne pathogens

From book: Biosensing Technologies for the Detection of

Pathogens - A Prospective Way for Rapid Analysis
 Microbial Detection

Microbiology analysis Molecular Detection

Polymerase chain reaction (PCR)
Molecular Detection and Identification
What is Polymerase chain reaction
(PCR)
 PCR is an exponentially progressing
synthesis of the defined target DNA
sequence in vitro. Thus, many target
sequence copy is produced.
 The PCR product calls amplicons or PCR
amplification products.
 It was invented in 1983 by Dr. Kary Mullis,
for which he received the Nobel Prize in
Chemistry in 1993.

Dr. Kary Mullis

 Theoretical Yield Of PCR
Theoretical yield = 2n x y
Where y = the starting
number of copies and
n = the number of thermal cycles

If you start with 100 copies, how many copies are made in 30
cycles?

2n x y
= 230 x 100
= 1,073,741,824 x 100
= 107,374,182,400
More Cycles = More DNA
Marker Number of cycles
0 10 15 20 25 30
 PCR : principle (1)
1
5' 3'

3' 5'
2

5' 3' Denaturation:

3' 5' 94 – 96°C

5'
3' 5'
3' Annealing:
3'
5' 3'
5' 50 – 65°C

1
5'

3' 5'
3' Elongation:
3 4 70 – 75°C
5' 3'

3' 5' Cycle 1

2
 PCR : principle (2)

1
5' 3'
5'
3'
5
3'
7
5'
3' 5'
3

4
5' 3'
5'
3'
6 8
3'
5'
3' 5'

Cycle 2
 PCR : principle (3)

5' 3'

3' 5'
5' 3'
3' 5'

5' 3'

3' 5'
5' 3'
3' 5'

5' 3'

3' 5'
5' 3'
5'

5' 3'
16
3' 5'
5' 3'
3' 5' Cycle 3
 Principle of PCR II
PCR : n Cycles
Initial Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6

After n Cycles = 2n Copies

 Exponential
Summary
microbiology and Weigh the Sample
25 g/ 225 ml
molecular analysis
Direct plating Enrichment media

Selected media

Identify

Biochemical test Molecular analysis

Polymerase chain reaction

 PCR process
Colonies
preparation Day 1-2

Day 3
DNA extraction 1 hour

Taq polymerase, Day 4

About
PCR mixture Primers, DNA 60 min
sample, dNTPs,
Buffer
Amplification

Detection using agarose gel

view under UV
 DNA extraction for bacteria

Break down the

cells wall and cell
membrane to
release the DNA

Cell growth

Cell harvest and lysis

DNA concentration DNA purification

5 DNA pellet
DNA purification
 Measurement of Nucleic Acids
Spectrophotometrically
 quantity
 quality
Fluorescent dyes
 gel electrophoresis

A260 1.0  50 g/ml

DNA
A260/A280 1.6 - 1.8
A260 1.0  40 g/ml
RNA
A260/A280 ~2.0
Spectrophotometric method
 Basic Components of PCR
 Template DNA (0.5 - 50 ng)
< 0.1 ng plasmid DNA, 50 ng to 1 μg gDNA for single
copy genes

 Oligonucleotide primers (0.1 – 2.0 μM):

A DNA polymer made up of a small number of
nucleotides
 dNTP’s (20 –250 μM)
 Taq polymerase (0.5 – 2.5 U/reaction)
 MgCl2 (1 – 5 mM) affects primer annealing and Taq Buffer
activity
Primers
 Buffer (supplied as 10X) ACTG
Working concentrations
dNTPs
KCL (10 – 50 mM) DNA template
Tris-HCl (10 mM, pH 8.3) Taq polymerase ++ MgCl2
NaCl2 (sometimes)
 Step 1:
Denaturation
dsDNA to ssDNA

Step 2:
Annealing
Primers onto template

Step 3:
Extension
dNTPs extend 2nd strand

Vierstraete 1999

Extension products in one cycle serve as template in the next

PCR amplification: thermo cycling protocol

1X 35X 1X
94oC 94oC
3 min 1 min 72oC
1 min
Initial denaturation 55oC
of DNA 45 sec

denaturation

extension
4oC

annealing
∞ hold
Example thermal cycler protocol used in lab:

Step 1 7 min at 94˚C Initial Denature

Step 2 45 cycles of:

20 sec at 94˚C Denature

20 sec at 52˚C Anneal
1 min at 72˚C Extension

Step 3 7 min at 72˚C Final Extension

Step 4 Infinite hold at 4˚C Storage

 Gel electrophoresis

 A technique for separating DNA /protein molecules of varying by

moving them through a block of agarose gel
 Analytical or preparative method

 Separates fragments with large range of molecular weights

 Driven by simple current and the

 fact nucleic acids are negatively charged

 Sample and tracking dye (TD) or loading buffer - used to keep

sample in the well and visualize run
 DNA is negatively charged.
 When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
 An agarose gel is used to slow the movement of DNA and separate by
size. H O 2
 

DNA

- +

Power Polymerized agarose is porous,

allowing for the movement of DNA
Scanning Electron Micrograph of
Agarose Gel (1×1 µm) 
How fast will the DNA migrate?

 strength of the electrical field, buffer, density of agarose gel

 Small DNA move faster than large DNA
gel electrophoresis separates DNA according to size

DNA

small
large

- +

Power
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
 Electrophoresis Equipment
Power supply

Gel tank Cover

Electrical leads


Casting tray

Gel combs
 Preparing the Casting Tray

Seal the edges of the casting tray and put in the combs. Place the casting
tray on a level surface. None of the gel combs should be touching the
surface of the casting tray.
 Pouring the gel

Allow the agarose solution to cool slightly (~60ºC) and then

carefully pour the melted agarose solution into the casting tray.
Avoid air bubbles.
When cooled, the agarose polymerizes, forming a flexible gel. It should
appear lighter in color when completely cooled (30-45 minutes).
Carefully remove the combs and tape.
 DNA

buffer     
wells

Anode
Cathode (positive)
(negative)

Add enough electrophoresis buffer to cover the gel to a depth of

at least 1 mm. Make sure each well is filled with buffer.
 Sample Preparation
 Mix the samples of DNA with the 6X sample loading buffer (tracking
dye).
 This allows the samples to be seen when loading onto the gel, and
increases the density of the samples, causing them to sink into the gel
wells.
6X Loading Buffer: 
 Bromophenol Blue (for
color)
 Glycerol (for weight)
 Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample. The
sample should sink into the well. Be careful not to puncture the gel with the
pipette tip.
 Running the Gel

Place the cover on the electrophoresis chamber, connecting the electrical leads.
Connect the electrical leads to the power supply. Be sure the leads are attached
correctly - DNA migrates toward the anode (red). When the power is turned on,
bubbles should form on the electrodes in the electrophoresis chamber.
Right way to label the gel on the paper
Species-specific of ATP 6 for porcine DNA detection

FIGURE 1. Species-specific of ATP 6 primer at 3.0% (w / v) agarose gel. Lane M: 100 bp DNA ladder; Lane
NC: negative control; Lane 1 chicken; Lane 2: duck; Lane 3: fish; Lane 4: buffalo; Lane 5: bull; Lane 6:
goat; Lane 7: pig; Lane PC: positive control (83 bp).
1

2 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

bp

10000

2500

1500
1000

500 368 bp

4 Fig. 2 Detection of toxR gene of Vibrio parahaemolyticus isolates from Negeri Sembilan by polymerase chain

5 reaction (PCR) electrophoresed on 1.5% (w/v) agarose gel. M, Molecular weight sizes (base pairs, bp) are indicated

6 by numbers on the left; lane 1-18: VPN1-VPN18

Detection of Salmonella spp. according to ISO6579:2002
*Muller-
25 g of sample +225 ml of buffered KauffmannTetrathionate-
Pre-erichment peptone water: homogenization Novobiocin Broth
*Rappaport-VASSILIADIS
Day 1 Salmonella Enrichment Broth
Incubation at 37oC ± 1oC for 18 ± 2 h

Selective 1 ml of homogenate to 10 ml 0.1 ml of homogenate to

enrichment MKTTn* incubation at 37oC ± 10 ml RVS incubation at
Day 2 1oC for 24 ± 3 h 41.5oC for 24 ± 3 h

Isolation on Plating out by 10 incubation at 37oC ± 1oC for 24 ± 3 h

selective agar  Xylose lysine desoxycholate agar (XLD)
Day 3  Other selective medium (BGA, chromogenic media)

Isolation and characterization of colonies on Nutrient agar and

incubate at 37oC ± 1oC for 24 ± 3 h

Confirmation Confirmation
Day 4-6
Detection of Salmonella spp.

Multiplex PCR was conducted by using 3 sets of primers:

i) ST11 (5’-GCCAA CCATT GCTAA ATTGG CGCA-3’) and ST15 (5’-
GGTAG AAATT CCCAG CGGGT ACTGG-3’) for detection of
Salmonella spp. targeting random sequence gene (429 bp) (Soumet et
al., 1999).
ii) ENTF (5’-TGTGT TTTAT CTGAT GCAAG AGG-3’) and ENTR (5’-
TGAAC TACGT TCGTT CTTCT GG-3’) for detection of S. Enteritidis
targeting SdfI gene (304 bp) (Alvarez et al., 2004).
iii) Fli15 (5’-CGGTG TTGCC CAGGT TGGTA AT-3’) and Typ04 (5’-
ACTGG TAAAG ATGGC T-3’) for detection of S. Typhimurium
targeting the fliC gene (620 bp) (Soumet et al.,1999).
Amplification of DNA was performed in 25 μl
components Volume per sample Concentration
DNA template 2 µl
5x PCR buffer 5 µl
dNTP 0.5 µl 10 mM
mM MgCl 2.5 µl 25 mM
Primers 0.5 μl 0.2 μM for ST11 and
ST15, 1.2 μM for Fli15,
Typ04, ENTF and ENTR
Taq polymerase 0.3 μl
Sterilled distilled water 14.2 μl
TOTAL 25 μl
 Detection of Salmonella spp.
M 1 2 3 4 5 6 7 8 9

bp
1500

700
620 bp
600
429 bp
300
304 bp

Figure 1. Gel electrophoresis of multiplex PCR of SdfI gene and fliC gene for identification of Salmonella
spp. (429 bp), S. Enteritidis (304 bp) and S. Typhimurium (620 bp), respectively. Lane M: 100 bp DNA
ladder; Lane 1 and 2: positive control of S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076,
respectively; Lane 3-8: representative positive samples; Lane 9: negative control.calibr
Summary
microbiology and Weigh the Sample
25 g/ 225 ml
molecular analysis
Direct plating Enrichment media

Selected media

Identify

Biochemical test Molecular analysis

Polymerase chain reaction

Identification of bacteria Identification of bacteria

(Pengenalpastian bakteria) (Pengenalpastian bakteria)

Combination technique for

confirmation/ kepastian