Master of Science in Molecular Biotechnology
REPORT ON ELECTIVE PRACTICALS IN FUNCTUNAL GENOMICS PART II:
SALMONELLA DETECTION TECHNIQUES AND IN SITU HYBRIDIZATION
Jhonny Correa1, Dr. Manfred Mielenz 2
1
Student of the Master of Science in Molecular Biotechnology (MN. 1972158). Center of Molecular Biotechnology. 2Practical
Supervisor. Institute of Animal Science. Department of Physiology and Hygiene. University of Bonn, Germany. January 8, 2007.
INTRODUCTION
Advances in science have always been closely
related to the development of technology.
Particularly, humanity have strongly benefited
from the fast growing number of techniques
registered in the area of genetics in the last two
decades, which have resulted in significant
improvements to several of the biotechnological
process that provide many of the products in
today’s markets. Interesting applications of gene
techniques can be illustrated with the methods
applied for the analysis of Salmonella (enteric
bacteria) and the in situ hybridization.
Detection and Quantification of Salmonella.
These bacteria may pass from the faeces of people
or animals to other people or other animals,
commonly by ingestion of Salmonella
contaminated food products. It usually results in a
serious gastrointestinal infection and reported
cases (in humans) currently reach alarming levels
in some regions worldwide and many countries
have adopted a Salmonella-free food position [1].
Consequently, careful analysis of food is always
required and so new methods; however, their
implementation involves meticulous evaluation
prior to their authorization.
The oldest method for Salmonella analysis is
Platting, which applies microbiology techniques
for in vitro culture to isolate the bacteria which is
subjected to biochemical tests to support its
identity. The procedure consider enrichment
periods in both non-selective and selective culture
media (e.g. peptone water and Rappaport-
Vassiliadis RV, or tetrathionate broth base TBS,
respectively) [2], followed by incubation in solid
media (e.g. Brilliant-Green Phenol-Red, BPLS,
Xylose Lisine Desoxycholate XLD, Desoxicholate
Citrate or Mannitol lysine crystal violet brilliant
green agars) [3] for Salmonella detection.
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Characteristic biochemical properties of enteric
bacteria such as the degradation of sugar
accompany by acid production (triple sugar iron
agar, or during oxidation/fermentation with
glucose, lactose, etc), motility or indole
production (SIM), nitrite [4] and hydrogen
sulphite production (lysin Eisen agar) [5], and
others, are then analyzed for the isolated
suspicious colonies. pH indicators (e.g. phenol
red: 7.2<pH<7.4; yellow<red<fuchsia) are used
in the culture media to show the acid production
resulting of the metabolism of carbohydrates
during the growth of the bacteria [6]. Useful
biochemical test kits (e.g. enterotube [7]) are
nowadays commercially available. They have
made the biochemical test much easier; however,
in combination with platting, the identification of
salmonella still takes long (3-5 days, depending
also on the sample matrix and its pre-treatments).
Therefore, the need for more rapid methods able
to monitor Salmonella, all along the food chain,
has become crucial [8].
After the successful application of the Polymerase
Change Reaction (PCR), many studies involving
the DNA analysis for Salmonella detection and
quantification have been conducted [9,10,11].
Particular attention has been paid to the DNA
isolation methods and to the design of specific
primers. Possible DNA extraction methods
include the boiling, alkaline lysis, magnetic beads
extractions (e.g. Dynabeads) or solid phase
extraction using prefabricated kits (e.g. IQ-Check
or Nucleosin) [12,13].
DNA extractions mini kids (e.g. QIAamp® DNA
Stool Mini Kit) have been especially designed by
including steps/products to eliminate PCR
inhibitors and producing high purity DNA and
RNA [14]. A technique that combines QIAamp
kid with multiplex PCR has shown to be efficient
for the detection/identification of Salmonella and
other enteric pathogenic bacteria from stool
samples [15].
1 Jhonny Correa
Master of Science in Molecular Biotechnology
End-point PCR methods include a final
confirmation phase, such as electrophoresis,
which requires a certain level of skill and which is
both laborious and time-consuming, thus limiting
the number of samples that can be analyzed. In
recent years, a method based on PCR with an
automatic confirmation phase has been developed.
This method, known as real-time fluorogenic 5_-
nuclease PCR, has been used to detect a number
of pathogenic microorganisms, including
Salmonella. The method uses the 5_-nuclease
activity of Taq DNA polymerase to hydrolyze an
internal fluorogenic probe for monitoring the
amplification of DNA targets [13].
Whatever the PCR technique, it requires the
availability of primers and probes that must be
selected according to very rigid conditions, which
cannot always be easily applied. The use of the
double-stranded DNA (dsDNA) binding dye
SYBR Green I for the detection of PCR products
has overcome this limitation by allowing real-
time PCR to be applied without the need for
probes linked to fluorescent molecules; protocols
that are already in use for classic PCR can thus be
used with only slight modifications [13].
In the absence of probes, the specificity of the
reaction is determined by the melting temperature
(Tm) of the amplicon obtained, defined as the
temperature at which 50% of the DNA amplicon
is in a double-stranded configuration. The Tm
depends on various factors, including the
concentration of the dsDNA, the amplicon length,
and the nucleotide sequence. However, the
effectiveness of this method depends on the
capacity of the extraction and purification
procedure to remove from the sample those
substances that inhibit the activity of polymerase
DNA [13].
Another DNA based method for analysis of
Salmonella is the Colony Blot. In this procedure,
bacterial colonies are transferred on membranes,
lysed in alkaline conditions, cellular debris is
washed, and the DNA on the blot detected with
radioactive markers or enhanced chemical
luminescence, ECL [2]. One type of ECL makes
use of the chemical property of the compound
luminol, which effectively decomposed on the
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presence of oxygen (O2), releasing light energy
[16]. With the luminol-ECL detection method,
the amount of a particular DNA in a sample can
be measure indirectly, if the target DNA is
labelled with a specific molecule (e.g. horse radish
peroxidase) that can, in certain conditions (e.g.
presence of H2O2), release oxygen [2].
In situ hybridization (ISH).
Hybridization of nucleic acid molecules has been
optimized to allow microscopic examination of
genes in situ. ISH have made possible to
determine the chromosomal location of a
particular gene fragment or gene mutation. This is
carried out by preparing a radiolabelled (or
fluorolabelled, FISH) DNA or RNA probe and
applying this to a tissue or chromosomal
preparation fixed to a microscope slide. Any
probe that does not hybridize the complementary
sequences is washed off and an image of the
distribution or location of the bound probe is
detected. Using tissue or cells fixed to slides it is
also possible to carry out in situ PCR [6].
Optimal hybridization conditions strongly depend
on the type and GC content of the hybrids. RNA–
RNA and RNA–DNA hybrids will require higher
hybridization temperatures than DNA–DNA
hybrids. In general, the relative strength of
different hybrids is RNA–RNA hybrids>RNA–
DNA hybrids>DNA–DNA hybrids [17].
Fluorescence labelled probes (FISH) has
demonstrated to be highly useful for the detection
of specific mRNA. In this case the tissues are not
exposed to high pH, so the chromosomal DNA
remains double stranded and cannot bind the
probe. Instead the tissue is gently fixed so that its
RNA is retained in an exposed form that can
hybridize when the tissue is incubated with a
complementary DNA or RNA probe. In this way
the patterns of differential gene expression can be
observed in tissues, and the location of specific
RNAs can be determined in cells [18]. FISH has
revealed sites of RNA processing transport, and
cytoplasmatic location [2].
2 Jhonny Correa
Master of Science in Molecular Biotechnology
An application of FISH is the analysis of the
mRNA coding for haptoglobin, protein which
concentration increase in bovine serum as a result
of injury, infection or disease [19]. For this
application, a possible probe would be a fragment
of labelled complementary RNA (cRNA)
corresponding to the haptoglobin-coding mRNA.
One of recommended fluorophore labeling is
dioxigenin (DIG) which is incorporated into the
probe during the transcription using DIG-Uridine
Triphosphate. Probe-target hybrids are usually
detected with an alkaline- phosphatase-conjugated
antibody either by a color reaction or by a
chemiluminescence reaction [20].
The purpose of this report is to discuss the results
obtained during the application of some
techniques (platting, real time PCR and colony
blot) used to analyze the Salmonella content in
chicken (stool) samples and for in situ detection of
haptoglobin in pig liver (FISH) as a practical
training for basic knowledge of molecular
biotechnologist students.
MATERIALS AND METHOD
Detection of Salmonella by platting.
Chicken stool (10 g) was combined with peptone
medium (90g) homogenized and incubated 20 h at
370C. The pre-enriched culture (0.1 and 1.0 mL)
was inoculated in RV (9.9 mL) and TBE media
(90 mL) at 370C. Both of these cultures were
separately inoculated after 24 and 48 h in the
selective media MLCB [21] (2 petris/ sample a
day) and XLD agar (2 Petris/sample a day), and
incubated at 370C. One of the colonies grown in
MLCB (black colony) was inoculated in different
biochemical testing media (TSI, Lysine,
Haraastgff, OF, OF-oil, OF-lactose, Nitrate).
Preparation of free bacterial DNA
and Colony count.
This practice was done using an enriched culture
(peptone-water) of Bacillus subtilis. Dilution
series (10-1, 10-2…10-7) were preparated (1 mL
culture + 9 mL of sterile 0.9% NaCl solution) and
bacterial suspensions 10-2-10-7 (1.5 mL each) were
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separately transfer to a reaction tubes (1.5 mL
capacity). Dilution series 10-4-10-7 (0.1 mL each)
were additionally spread out on RV and TBS in
rich agar (1 petri per dilution) and incubated at
370C (colony growth was evaluated 24 h later).
Bacterial dilutions in reaction tubes 10-2,10-4 and
10-5 were centrifuge (6000 g, 40C) 20 min;
immediately placed on ice and the liquid phase
was removed by pipetting. Bacteria pellet was
washed with pre-cooled 1X PBS (500 µL/ tube;
(6000 g, 40C, 10 min); samples were placed on ice
and the buffer was removed. Cells were lysed by
re-suspending the pellet in ddH2O (50 µL; mixing
thoroughly for 30 s) followed by a water bath
(950C) 10 min. Tubes with DNA in solution were
cooled down on ice (5 min), spined down briefly
and freeze at -20oC.
DNA extraction by the DNA stool mini kit and
analysis with real time PCR.
This practice was performed using chicken stool
samples purposely contaminated with Salmonella.
Bacteria pellet, on reaction tubes (2 mL capacity),
had been prepared by dilution of an enriched
cultured, at concentrations 10-2, 10-4. DNA was
released from the bacterial dilutions on the
reaction tubes by suspending the pellets in ASL
buffer (1.4 mL, vortex continuously for 1 min),
followed by a water bath (700C) during 5 min.
The samples was mixed 15 s, centrifuged (16 000
g) 1 min and the supernatants (1.2 mL, containing
the DNA) were separately transferred into a fresh
2 mL reaction tubes.
PCR inhibitors were removed with InhibitEX-
tablets which were added (1/sample) to the
supernatants, the mixtures were vortex (1 min),
incubated (1 min) and centrifuged (16 000 g, 5
min); supernatants were transferred into fresh 1.5
mL reaction tubes and centrifuge again (16 000 g,
5 min). Possible associated proteins to the
bacterial DNA were eliminated by adding the last
supernatants (200 µL) into a 1.5 mL reaction tube
containing Proteinase K solution (15 µL) with
subsequent addition of AL buffer (200 µL) and
vortex (15 s); the mixtures were heated (700C, 10
min), spined down, ethanol (200 µL) was added,
mixed and spined down.
3 Jhonny Correa
Master of Science in Molecular Biotechnology
DNA in the samples was purify by anion
exchange transferring the lysate solutions into
spin-columns (QIAamp) lying in fresh 2 mL
reaction tubes and centrifuging (16 000 g, 1 min);
impurities were eluted in two steps, with AW1
buffer (500 µL) and AW2 buffer (500 µL),
followed by centrifugation (16 000 g, 1 and 3 min
respectively). The DNA was then eluted from the
spin-column (in fresh reaction tubes) with AE
buffer (200 µL) after incubation (RT, 1 min) and
the DNA solution was stored at -200C. The
samples were analyzed next day by real time PCR
using SYBR Green I to detect Salmonella gene
sequence (InvA, 172 bp [22]) under the following
thermal cycling conditions: 95°C (10 min) and
[94°C (30 min), 55°C (30 sec) and 72°C (30 sec)]
33 cycles.
Colony blot.
A mixture of cells (Salmonella enteritides,
Echerichia coli and Staphylococcus aureous) was
spread out on peptone media in a Petri dish and
incubated (37oC) overnight plus 2 h at 4oC. The
dish was brought out of the refrigerator and a
square piece of blotting membrane placed on the
colonies until it got completely wet; its location
was marked, the membrane was removed and the
Petri dish was incubated again.
Cells attached to the membrane, were lysed by
placing it (colony site up) on two layers of filter
paper soaked with the following solutions 0.5 M
NaOH (2 min), 0.5 M NaOH (5 min), 1 M
Tris/HCl (pH 7.5, 15 min), and twice 2X SSC (1
min each). Colony blots on filter paper were air
dried, DNA fixed under UV-light (312 nm, 5min)
and stored overnight wrapped in filter paper and
aluminum foil at -200C.
DNA impurities on the blot were removed by
placing it in the pre-heated (420C) hybridization
tube (3x10 cm, dxh) containing 5X SSC (5 mL,
also pre-heated) followed of incubation (420C, 10
min) in the rotisserie oven. The SSC buffer was
removed, pre-hybridization buffer (45 mL) was
added and the system incubated (42oC, 15 min) in
the rotisserie oven. DNA labelled probe (120 µL,
prepared as described bellow) was added in to the
buffer and allowed to hybridize the Salmonella
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DNA on the blot by incubating (42oC) the system
overnight in the rotisserie oven. Labeling probe in
excess was removed by discarding the
hybridization buffer and washing (at 42 oC) with
5X SSC (50 mL, 5 min), urea-containing buffer
(50 mL, 20 min) and was buffer (50 mL, 20 min),
2X SSC buffer (50 mL, 5 min) and was buffer (50
mL, 5 min). The blot was placed on towel paper
in a plastic bag and detection of the Salmonella
DNA was performed by adding the detection
reagent (containing H2O2 and luminol) directly to
the blot and registering the signal generated
(during 30 min) in a film.
A solution (10 ng/µL) of a primer (172 bp) design
to detect InvA Salmonella gene sequence [22] was
used as a probe and placed in two 1.5 mL reaction
tubes (10 µL in each), denatured in a water bath
(1000C, 5 min), cooled down on ice (5 min) and
spined down briefly. The ssDNA probes were
labelled with horse radish peroxidase (HRP)
enzyme (10 µL/tube), which was added to the
reaction tube, spined briefly; glutaraldehyde
solution (10 µL/tube) was then added and spined
down briefly. The reaction tubes were incubated
(37oC, 10 min), 80% glycerol was added and the
labelled probes were stored (-200C) overnight.
Detection of specific mRNA by
in situ hybridization.
A small piece of a frozen pig liver was sliced (12
µm, T= -40C) in a cryomicrotome. Two tissue
slices were put on two different slides, which were
then placed into a glass chamber, 4% PFA-PBS
(paraformaldehyde-1X phosphate buffer solution,
50 mL, RT) and intracellular protein cross-linking
reactions allowed to proceed during 15 min. PFA
in excess was washed twice with 1X PBS (5 min
each, RT) and the tissues were dehydrated by
exposing the tissue on the slides for 5 min to each
of the following fresh ethanol solutions in ddH2O
50%, 70%, 90% and 100%. The slides were
allowed to go dry (370C) in 30 min and stored 16
h at -800C.
Next day, the slides were re-hydrated by
subsequent incubation (RT, 5 min) in each of the
4 Jhonny Correa
Master of Science in Molecular Biotechnology
following fresh-prepared ethanol solutions 100%,
90%, 70%, 50%, twice in 1X PBS buffer, 10 min
in a mixture of 0.25% acetic anhydride with 0.1 M
triethanol amine (125 µL plus 50 mL,
respectively), and 10 min in 2X SSC. One of the
slides was hybridized with sense probe (50 µL)
and the other one with anti-sense probe (50 µL);
both of the probes referring to the parcial mRNA
for haptoglobin of Bos taurus (302 bp) [23] and
DIG labelled. The slides were covered with a slip
and incubated (520C) overnight in a humid
chamber.
After hybridization, the excess of probe was
removed by subsequent incubation (450C, 10 min)
of the slides in the preheated (450C) solutions of
2X SSC (twice), a mixture 1:1 formamide/2X
SSC (twice), 0.2 X SSC (twice, RT, shaking).
Unlabelled RNAs (ssRNA) in the tissue were
digested by incubation (370C, 30 min) in a
solution 50 µg/mL RNase A and 1000 µg/mL
RNase T1. Excess of RNase was removed by
incubation (RT, 10 min) in 2X SSC (50 mL, three
times).
To avoid background signals, the slides were
incubated in maleic acid buffer MAB (50 mL) and
1% blocking solution (45 mL MAB+ 5 mL 10%
blocking reagent stock solution, 30 min). The
slides were then taken off, the excess of blocking
solution was carefully removed and the tissue
sections were circumvented with a silicon pen.
Inmuno detection was performed with Anti-DIG
Fab-fragments (100 µL, labelled with 1:500
alkanine phosphatase/1% blocking solution) by
incubation (RT, 2 h) in a humid chamber. The
excess of antibody was washed by incubation
(RT, 15 min) in a solution of 10% Tween in MAB
(1:99), three times in MAB, 10 min in substrate
buffer and overnight (in the dark) in a mixture of
NBT (75 mg/mL in 70% DMF), BCIP (50 mg/mL
in DMSO) and substrate buffer (225 µL, 175 µL
and 50 mL, respectively).
The slides were incubated (RT, 10 min) in TE-
buffer, taken off from the chamber, Kaiser’s
glycerin (2 drops) was added on the tissue section,
it was protected with a cover slip and the tissue
was evaluated under the microscope.
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RESULTS AND DISCUSSION
Detection of Salmonella by platting.
Evaluation by platting of selective enriched media
for colonies morphology of grown bacteria
showed that non of the organisms in plates with
XLD did present the particular features expected
for Salmonella colonies (Figure 1a); however, in
MLCB there were suspicious colonies (black
color, figure 1b), so a corresponding pure
representative was submitted to biochemical test.
There were no differences between 24 and 48 h
(Petri) cultures.
a b
Figure 1. Plates used as patterns to compare results for
Salmonella detection. a) In XLD media Salmonella
colonies (e.g. the one signaled with an arrow) look pink
and the media become also pink. b) In MLCB media
colonies look black and the surrounding media turns
lightly yellow.
Qualitative biochemical results (Table 1) shows
that the colony analyzed share with salmonella the
metabolic abilities of producing H2S (TSI+ and
SIM+), degradation of lysine, motility (SIM+),
facultative aerobics (O/F+ and O/F-oil+), nitrite
production (GRISS ILOSVAY’S reagent) and
proteons positive (Harastoff). These similarities
explain why this bacterium could grow in one of
the selective media (MLCB) showing related
colony features.
Contrary to most of the Salmonella spp., the
analyzed bacteria do metabolize lactose; although
there are some Salmonella strands that producer β-
galactosidase, as there were no suspicious
colonies in XLD agar, there is low probability
(<3%) that this is a Salmonella spp.
5 Jhonny Correa
Master of Science in Molecular Biotechnology
Table 1. Biochemical test results.
Media
Color Change
Decision RS*
Inicio-Final
TSI + +
Lysine + +
Harastoff + +
SIM + +
O/F-oil + +
O/F + +
OF-Lac + -
GRIESS + +
*Reported results for Salmonella24.
This detection method is slow, but very sensible
since after pre-enrichment period, if there was
only one bacterium in the sample and it could
poorly multiply, the observation of a single
suspicious colony in the selective agars will be
notice. Moreover, supported with biochemical
tests, it is usually possible to concluded positive
results very easy.
Preparation of free bacterial DNA
and colony count.
DNA from B. subtillis was isolated; however, the
results were not confirmed. Density (Č), colony
forming units per milliliter (CFU/mL), determined
for B. subtillis in the enriched culture (Table 2)
resulted similar in both RV and TBS media.
Despite the relatively close density obtain in both
of the media, the values used to calculate the
density in TBS data of dilution 10-6 were out of
the reliable range for quantification (20-300
colonies), which means the bacteria concentration
was on the quantification limit. If the average
value is calculated, the resulting density of B.
subtillis in the enriched culture is 2.3x107
CFU/mL.
When using this quantification method, it should
be kept in mind that it assumes that each colony is
derived from a single cell, which may not always
be the case, since colony clumps were observed in
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the dishes. Moreover, it is also possible, that
suboptimal culture conditions may cause poor
growth, thus leading to an underestimation of the
true density.
Table 2. Data registered to detect the concentration of
B. sublillis by colony count.
Dilution Colonies-RV Colonies-TBS
10-5 128 25
10-5 173 35
-6
10 26 4
10-6 26 3
Č* (CFU/mL) 1.604x107 3.045x107
*Calculated as in [2]
DNA extraction by the DNA stool
mini kit and real time PCR.
DNA was extracted and quantify by real time
PCR. Bacteria detection/density in the samples
was examined by comparison with standards
prepared by the instructors. Computer analysis of
the PCR results (Table 3) showed a linearity of
0.990, a threshold of 203.409 was used and the
slope for the standard curve (Figure 2) had a value
of -3.7 Ct/copy. Based on these data the program
calculated the amount of copies in the dilutions.
The number of DNA copies detected in dilutions
10E-2 show reproducible values (i.e. 4.03E+01 vs
4.29E+01 and 1.68E+02 vs 3.01E+01) for the same
analyst. Considering that there is no consistency
for bacterial solutions more diluted (i.e. 10E-4-
10E-7), it is possible to say that these number of
copies are under the quantification limit for this
method.
However, in view of that the analysts were not
experts; the detection would be possible even at
the lowest concentration assayed (10E-7). Thus,
as this dilutions were also analyzed in PCR-AGE
(see last page) a comparison of the methods lead
to the conclusion that the sensitivity of Salmonella
by real time PCR (dilution 10E-7) would be about
100 fold higher than that of the PCR-AGE
(dilution 10E-5).
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Master of Science in Molecular Biotechnology WS 2006/2007

Table 3. Results of samples analyzed in real time PCR .

Sample Ct (dR) Quantity (copies)
Standard 10E-1* 33.42 1.00E+01
Standard 10E-1* 33.55 1.00E+01
Standard 10E-2* 30.07 1.00E+02
Standard 10E-2* 29.44 1.00E+02
Standard 10E-3* 26.36 1.00E+03
Standard 10E-3* 26.08 1.00E+03
Standard 10E-4* 21.89 1.00E+04
Standard 10E-4* 22.28 1.00E+04
Standard 10E-5* 18.98 1.00E+05
Standard 10E-5* 18.81 1.00E+05 Figure 3. Dissociation curve for samples in Table 3
Standard 10E-6* 14.74 1.00E+06 during real time PCR analysis.
Standard 10E-6* 14.68 1.00E+06
Standard 10E-7* 11.8 1.00E+07 The analysis of the dissociation curve (Figure 3)
Standard 10E-7* 11.03 1.00E+07 shows that amplicons have very similar
Dilution 10E-2 31.23 4.03E+01 denaturation temperature (93.5-95.0 oC), which
Dilution 10E-2 31.13 4.29E+01 means that the specificity of the InvA base primer
Dilution 10E-2** 28.93 1.68E+02 and so the selectivity was/were excellent, which
Dilution 10E-2** 28.89 1.73E+02 agree results reportes in [22]. The variations in
Dilution 10E-4 38.59 4.14E-01 that narrow range (and not a unique maximum)
Dilution 10E-4 ND*** ND*** would be because the concentration of the
Dilution 10E-4** ND*** ND*** amplicons in the samples is different.
Dilution 10E-4** ND*** ND***
Dilution 10E-5* 36.99 1.11
Samples were no signal was detected (ND, Table
Dilution 10E-5* ND No Ct
3) did not behaved logarithmically during the
Dilution 10E-5* 27.08 5.33E+02
Dilution 10E-6* 31.7 3.01E+01
amplification (Figure 4), which reveals the
Dilution 10E-7* 35.49 2.84 weakness of the real time PCR, since its
*QIAamp mini kid worked by the instructors or a college** sensibility and quantification limits depend on a
***ND=Not detected. logarithmic amplification of the DNA. Based on
this argument, it is reasonable to think that ND
samples did experiment some kind of inhibition
during the stage of PCR amplification or that for
some reason the DNA was lost during the
treatment with the QIAamp mini kit.

Figure 2. Curve for standards (square spots) and

dilution samples (tringles signs) in Table 3.
Figure 4. Amplification plots of samples in Table 3
during the real time PCR analysis.

Elective Practical in Functional Genomic (Part II) 7 Jhonny Correa

Master of Science in Molecular Biotechnology
Figure 4 was possible since contrary to all the
other PCR techniques, real time PCR allows
monitoring the results as they are collected
without the need to stop the analysis.
Colony blot.
The developed film showed several black spots
and the film was faithfully photocopied. The
Petri corresponding to the blot (which had been
re-incubated) was overlaid to the photocopy to
compare (Figure 5). It could be observed that
after re-incubation, colonies on the plate, which
had been adsorbed on the blot, could grow again
and one type of colony (the one corresponding to
Salmonella) did match perfectly (100%) all of the
spots on the blot. Colonies of other bacteria
(Echerichia coli and Staphylococcus aureous.)
were not detected using the InvA complementary
fragment.
Figure 5. Re-incubated Salmonella colonies did match
perfectly to black spots resulting of the specific ECL
detection of InvA gene fragment for Salmonella colonies
on the blot. Even very small colonies can be detected
(e.g. the one singed with the arrow).
The high sensitivity of ECL using the pair
Luminol-HRP can be perceived by looking that
even the size of the smallest colonies that resulted
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developed (e.g. the one marked with an arrow in
Figure 5), which is an advantage in case the
bacterium to be detected grows slowly.
Detection of specific mRNA by
in situ hybridization.
Comparison of slides with the liver tissue (i.e. anti
vs the sense labelled probes) could be seen could
be done at nicked eye (Figure 6). From these
results, it can be inferred that hybridization of the
haptoglobin coding mRNA with the sense probe is
not stable, so this probe could be extracted from
the tissue during the washing steps.
a b
Figure 6. Appearance of pig liver tissue slides
hybridized in situ with Anti-DIG labelled anti (a) and
sense (b) probes after detection with alkaline
phosphatase.
Tissue labelled with the anti probe was evaluated
under the microscope (Figure 7) were brown-red
spots did appear spread out the tissue following
some distribution pattern as the expected for
hepatocytes (see additional picture, answer to
questions section). This indicates that particular
sections on these cells were selectively detected.
In fact, further magnification (Figure 8) allowed
seeing that stronger stained areas belong to the
hepatocyte nucleus (sphere shaped), which means
that the higher concentration of the mRNA coding
for haptoglobin in these cells is inside the nucleus,
however, at this magnification it is not possible to
determine any specific distribution. Moreover, it
is also possible that the nucleus region present
some background problem.
8 Jhonny Correa
Master of Science in Molecular Biotechnology
Figure 7. Low power magnification of the liver tissue.
Figure 8. Magnification of the liver tissue with a higher
power.
Poor detection in the cytoplasm could be due to a
very low concentration of the mRNA which
would suggest that this mRNA once in the
cytoplasm is fast transcripted, degraded, or
somehow complex in a way that could not be
hybridized by the probe.
Another interpretation of the low signal registered
from the cytoplasm would be that it is background
staining (or both possibilities together). However,
since it could be seeing that nucleus closer to the
tissue surface present better staining (which could
be because the probe was in too low
concentration, did required more time to diffuse
into the tissue or other facts that resulted in a poor
access to the internal cells) the first option is more
acceptable.
Elective Practical in Functional Genomic (Part II)
WS 2006/2007
CONCLUSION
The PCR methods are faster, more specifics and
more sensitive than platting, however there is
always the risk of losing DNA during its
extraction since usually several steps are required;
if a protocol is establish for determined kind of
sample, there should be no problem to relay on the
results of this method, a great advantage or the
PCR methods is that several steps can be
automated, therefore allowing the analysis of a
greater number of samples. As bacteria have to be
dead in order to extract the DNA, PCR methods
represent a more safe method for the analyst. On
the other hand the platting method is cheaper.
A basic requirement for the PCR methods to work
properly if that the target DNA reach the
amplification step in optimal conditions, so for a
successful detection/ quantification it is
fundamental to know if the sample to be analyzed
has PCR inhibitor. If the sample is of certain
degree of purity (as an enrichment) it is possible
that a DNA extraction using the boiling method
will give DNA of the required purity. On the
other hand, if the sample to be analyzed include
different components (e.g. chicken stool), it is
fundamental to include steps for removing
possible inhibitors (e.g. polyphenols) but
recovering as much DNA as possible.
The degree of specificity in the detection of the
colony blot method depends on probe and the
InvA fragment used was 100% effective to detect
Salmonella enteritidis.
Probes designed based on a specific sequence of
mRNA in a specie (e.g. coding for hoptoglobin in
Bos taurus) would be use to hybridize strong
enough the corresponding mRNA in a different
specie (e.g. pig liver), so they can be detected.
Thus it could be determined that the concentration
of mRNA coding for haptoglobin is higher in the
nucleus than in the cytoplasm.
Aknowledgement
I am grateful with the laboratory instructors Dr
Manfred Mielenz, Isabella Israel, Inga Hofs and
all the staff of Department of Physiology and
Hygiene for the practical hints they did show.
9 Jhonny Correa
Master of Science in Molecular Biotechnology WS 2006/2007

REFERENCES
and Laura Toti. Evaluation of DNA
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J. S. Karns, J. S. Van Kessel, B. J. McCluskey Extraction Methods for Use in Combination
and M. L. Perdue. Prevalence of Salmonella with SYBR Green I Real-Time PCR To Detect
enterica in Bulk Tank Milk from US Dairies Salmonella enterica. Applied and
as Determined by Polymerase Chain Reaction. Environmental Microbiology, June 2003, p.
J. Dairy Sci. 88:3475–3479 3456–3461
2 14
MBT121 Funtional genomics scripts. http://www1.qiagen.com/resources/info/qiagen
3
John D. Perry, Michael Ford, Jeffrey Taylor, _purification_technologies_4.aspx?
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Amanda L. Jones, Roger Freeman, and http://www1.qiagen.com/literature/qiagennews
Frances K. Gould. ABC Medium, a New /0400/Detection%20of%20pathogenic.pdf
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Chromogenic Agar for Selective Isolation of http://en.wikipedia.org/wiki/Luminol
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Salmonella spp. J Clin Pathol 2002;55:286– http://www.biocompare.com/technicalarticle/
288 859/The-DIG-System-—-Nonradioactive-
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Merck Microbiology Manual. 12 edition. 303, And-Highly-Sensitive-Detection-Of-Nucleic-
372, 373, 434, 471, 472, 487 Acids-from-Roche-Applied-Science.html
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http://www.sifin.de/deutsch/produktinfo/lysin- Albert, B.; Johnson, A. Et al. Molecular
eisen-agar-tn1155.pdf Biology of the Cell. 2002. Fourth edition, p
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K. Wilson and J. Walter. Principles and 497-8
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Techniques of Biochemistry and Molecular http://www.lifediagnostics.com/2410-7%20Inse
Biology. 2005. Cambridge University Press. rt.pdf
6th Edition. p 83, 272-3 20
http://www.biocompare.com/archive.asp?pagey
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http://www.mc.maricopa.edu/~johnson/labtools ed=20&itemid=50927
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/Dbiochem/ent.html http://www.sifin.de/english/produktinfo/mlcb-
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http://www.bio-rad.com/cmc_upload/Techno agar-tn1266.pdf
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logy_ Spotlights/ 97246 /Salmonella_GB.pdf http://www.bfr.bund.de/cm/222/evaluation_o
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Twelve hour real-time PCR technique for the f_salmonella_spp._specific_primer_sets_for_t
sensitive and specific detection of Salmonella he_validation.pdf
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in raw and ready-to-eat meat products Jay L. http://ctd.mdibl.org/detail.go;jsessionid=F61
E. Ellingson, Jennifer L. Anderson, Steve A. FF12B41736807F07275355AB28DA1?type=s
Carlson and Vijay K. Sharma. Molecular and eq&acc=AJ271156
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Cellular Probes 18 2004 51-57 Madigan, M.; Martinko, J. and Parker, J.
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Seo K. H.; Valentin-Bon I. E.; Brackett R. E.; Brock Biologia de los Microorganismos. 2004.
Holt P. S.; Rapid, specific detection of 10th editition. p 377-8
Salmonella Enteritidis in pooled eggs by real-
time PCRJournal of Food Protection 2004 67
5 864-869
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A.T. Csordas, J.D. Barak, M.J. Delwiche
Comparison of primers for the detection of
Salmonella enterica serovars using real-time
PCR. Letters in Applied Microbiology 2004
39 187-93
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Wang X.; Jothikumar N.; Griffiths M.W.
Enrichment and DNA Extraction Protocols for
the Simultaneous Detection of Salmonella and
Listeria monocytogenes in Raw Sausage Meat
with Multiplex Real-Time PCR Journal of
Food Protection. Vol. 67 No. 1 2004 189-92
13
Dario De Medici, Luciana Croci, Elisabetta
Delibato, Simona Di Pasquale, mma Filetici,

Elective Practical in Functional Genomic (Part II) 10 Jhonny Correa

Master of Science in Molecular Biotechnology WS 2006/2007

Answer to questions in the MBT121 Funtional genomics scripts.

1. Experiments involve in the detection of Salmonella.

2.3x107 CFU in 1 mL of peptone water.

Detection limit of the PCR

a. Heating method: 10-5-10-6

b. Directly extracted: 10-7
c. The PCR methods are faster, more specifics and more sensitive than platting, however there is
always the risk of losing DNA during its extraction since usually several steps are required; if a
protocol is establish for determined kind of sample, there should be no problem to relay on the
results of this method, a great advantage or the PCR methods is that several steps can be
automated, therefore allowing the analysis of a greater number of samples. As bacteria have to
be dead in order to extract the DNA, PCR methods represent a more safe method for the analyst.
On the other hand the platting method is cheaper.

The detection using the InvA gene (104) is approximately 105 times more sensible than the plating
method (109).

The DNA preparation using the boiling method (end point PCR) is less sensitive but I think it is
because of the detection method and not because of the DNA extraction method. It would had be
interesting to had detected on both real time and ethidium bromide (AGE) amplified samples
which DNA had been extracted by the same method, so the DNA extraction method could had be
comparable regarding to the sensitivity.
Yes, I did read reference [13] were for methods for DNA extraction (boiling, alkaline lysis,
Nucleospin, and Dynabeads DNA Direct System I) were compared (and the detection was for
real time PCR, like us). It is interesting that they found no different when detecting Salmonella
and decided to work with boiling point because it is rapid and simple.

The colony blot did show a specificity of 100% in my experiment.

I think colony blotting is the most reliable of the detection method we did use. However, real time
PCR have the advantages that is faster, the pretreatment is easier and that many steps can be
automated, I really think the PCR method is the future.

I think the real time PCR will play a fundamental key for future analysis of foodstuff and other
environmental samples in future because its high detection limit will reduce the enrichment
periods so the results will be given faster. And maybe one day using this technique the detection
of pathogens would be performed without the need of enrichment periods, which is currently a
disadvantage of all the methods.

Elective Practical in Functional Genomic (Part II) 11 Jhonny Correa

Master of Science in Molecular Biotechnology WS 2006/2007

2. In situ hybridization.

mRNAs are transcript with the sense sequence so they can only hybridize the anti probe.

a b hepatocyte

Figure 9. Images of rabbit liver stained with haematoxylin and eosin (H&E). a) Low magnification. The
best indication of a liver lobule are the large central veins and the strands/sheets of hepatocytes, which seem
to radiate out from the central veins. b) High magnification. The portal triads: portal vein, hepatic artery
and bile duct. One hepatocyte is indicated in a circle. No background problems in this staining, looks ok
since this is a stain of general applications.

Considerations to choose the optimal hybridization temperature. Type of hybrid (DNA-DNA,

RNA-RNA, DNA-RNA). GC and AT(U) content. Melting temperature, degree of homology
probe-target sequence and the hybridization buffer. Detailed formulas in case of using DIG
labelled probes at: http://www.roche-applied-
science.com/PROD_INF/MANUALS/DIG_MAN/dig190-192.pdf

Elective Practical in Functional Genomic (Part II) 12 Jhonny Correa

Master of Science in Molecular Biotechnology WS 2006/2007

EXTRA INFORMATION PROVIDED BY THE INSTRUCTOR.

Elective Practical in Functional Genomic (Part II) 13 Jhonny Correa