Molecular biology assignment.

“Detection of Botulinum toxin using ELISA and BoNT gene detection by PCR and
Southern blot hybridization.”
Objective:
“Detection of Clostridium Botulinum toxins using molecular and immunological methods.”

Outcome:
To explain the different types detection methods of botulinum toxins and the detection of
botulinum gene (Bot) using molecular techniques like PCR, Southern blot, etc. Also, to confirm
the presence of botulinum toxin in the samples using ELISA.

Introduction:
Clostridium botulinum
Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human
pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic
spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum,
and Clostridium baratii.
Group I strains of Cl. botulinum are referred to as ‘proteolytic’ and may produce toxin types A,
B or F. They are widely distributed in nature, being found in a variety of raw foods, and produce
highly heat-resistant endospores.
Group II strains are referred to as ‘non-proteolytic’ and produce toxin types B, E or F. They are
able to grow at temperatures as low as 3˚C. They are widespread in nature, but type E strains are
especially common in the marine environment. There has been much concern that these bacteria
may produce toxin in refrigerated processed foods without apparent spoilage. The spores of
Group II Cl. botulinum are much less heat-resistant than those of Group I strains.

Detection and identification of botulinum neurotoxin:

Detection of toxin-producing clostridia in the patient confirms the diagnosis. ELISA is the most
commonly applied immunoassay format in the detection of botulinum neurotoxins. Molecular
techniques targeted to the neurotoxin genes are ideal for the detection and identification of C.
botulinum.

Methodology:
Rapid detection by ELISA:
ELISA-based methods have been developed for examining food samples for botulinum toxins.
The neurotoxin in a sample binds to a solid test matrix that is usually pre-coated with polyclonal
or monoclonal capture antibodies against one or more toxins (sandwich ELISA). A sensitive and
specific ELISA is used to detect C. botulinum neurotoxins A, B, E, and F. The assay uses toxin
type-specific polyclonal antibodies to capture the toxin and digoxigenin (DIG)-labeled toxin
type-specific polyclonal antibodies as secondary antibodies. These DIG-labeled secondary
antibodies are then detected by anti-DIG antibody conjugated to horseradish peroxidase. This
enzyme is then detected using a chromogenic substrate.

ELISA:
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for
detecting and quantifying substances such as peptides, proteins, antibodies and hormones. In an
ELISA, an antigen must be immobilized on a solid surface and then complexed with an antibody
that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme
activity via incubation with a substrate to produce a measureable product. The most crucial
element of the detection strategy is a highly specific antibody-antigen interaction.
ELISAs are typically performed in 96-well plates, which passively bind antibodies and proteins.
A detection enzyme or other tag can be linked directly to the primary antibody or introduced
through a secondary antibody that recognizes the primary antibody. The most commonly used
enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). The choice of
substrate depends upon the required assay sensitivity and the instrumentation available for
signal-detection.

Basic procedure:

 The key step is the immobilization of the antigen of interest, can be accomplished by
direct adsorption to the assay plate or indirectly via a capture antibody that has been
attached to the plate.
 The antigen is then detected either directly (labeled primary antibody) or indirectly
(labeled secondary antibody).
 The most powerful ELISA assay format is the sandwich assay. This type of capture assay
is called a “sandwich” assay because the analyte to be measured is bound between two
 primary antibodies (the capture antibody and the detection antibody). The sandwich
format is used because of its sensitivity.

Molecular Detection of Clostridium botulinum in biological samples:

DNA-based detection methods include PCR and Southern blot hybridization, both of which are
sensitive and specific and are rapid. The molecular detection techniques are based on the
detection of the botulinum neurotoxin gene (bot) in the sample.

Polymerase chain reaction:

PCR protocols are based on ‘bont’ genes detection, which determine active components of
botulinum neurotoxin (BoNT). There are 3 different types of PCR used for the ‘bont’ gene
detection. Multiplex PCR for detecting several toxotypes simultaneously and Real-time PCR
using SybrGreen dyes and TaqMan molecular probes. Real-time or quantitative PCR is useful in
studies of gene expression; specifically differential expression of genes under different
environmental conditions or for comparative studies among different organisms. RT-PCR is used
to study the toxin gene expression in C. botulinum serotypes A, B, E, and F. For direct detection
of the gene from the sample, Nested PCR is used. The use of nested PCR enables to shorten time
of enrichment in culturing process or detection of this pathogen in sample.

Background:
The polymerase chain reaction (PCR) allows for the selective copying (i.e. amplification) of a
defined section of DNA within a DNA sample called the template. Commonly used templates
are genomic DNA, mitochondrial DNA, and complementary DNA (cDNA). PCR was described
by Kary Mullis in 1980, is based on the principle of enzymatic nucleic acid replication and is
applied for fast and easy amplification of DNA.
Most PCR protocols involve two primers (single stranded DNA oligonucleotides of about 25
nucleotides long) which, upon annealing to the template, dictate which portion of the template is
amplified. Also included in PCR reactions are the DNA nucleotides (i.e.dNTPs) and a mixture of
reagents is prepared to produce a reaction buffer, including magnesium chloride, Taq
polymerase, and the DNA sample (200 and 800 bp). This mixture is placed in a thermocycler,
which is programmed for a series of cycles depending on the microorganism and the primers
used.
General protocols of PCR:
PCR involves three basic steps:
1) DNA denaturation:
Using heat (~94 °C) to compromise the hydrogen bonds which keep the opposing strands
together. After an initial “hot start” of about three minutes, denaturation steps often only require
5 – 30 seconds.
2) Annealing:
Annealing of the primer to the complementary DNA strand. Annealing involves a cooler
temperature depending on the length of the primer, this step often occurs between 50 and 65 °C
for 30 - 120 seconds.

3) Extension:
Extension of the bound primers involve the thermal stable DNA dependent DNA polymerase
binding to the 3’ end of the primer and beginning to incorporate nucleotides dictated by
complementation with the template. Polymerases always add onto 3’ ends. This is referred to the
5’ to 3’ direction. Extension routinely occurs at 68 - 72 °C for one minute per kilobase (kb) of
template being amplified. After amplification, the products are separated by gel electrophoresis.
Simple schematic steps of PCR:

The specificity of PCR is confirmed by combining PCR with bot gene-specific probe
hybridization or by DNA sequencing of the PCR product. The PCR products are detected in
agarose gel electrophoresis and southern blot hybridization.
Agarose gel electrophoresis:
PCR products are analyzed in 1% agarose gel, Tris-acetate buffer and stained with ethidium
bromide. The electrophoresed products are visulized by UV- light source. Molecular size
markers are used to determine the molecular weights of the PCR products.
Background and general protocol:
Agarose gel electrophoresis separates DNA fragments according to their size. Typically, a DNA
molecule is digested with restriction enzymes, and the agarose gel electrophoresis is used as a
diagnostic tool to visualize the fragments. An electric current is used to move the DNA
molecules across an agarose gel, which is a polysaccharide matrix that functions as a sort of
sieve.
The agarose gel is a cross-linked matrix that is like a three-dimensional mesh or screen. The
DNA molecules are pulled to the positive end by the current, but they encounter resistance from
this agarose mesh. The smaller molecules are able to navigate the mesh faster than the larger one,
so they make it further down the gel than the larger molecules. This is how agarose
electrophoresis separates different DNA molecules according to their size. The gel is stained
with ethidium bromide to visualize the DNA molecules resolved into bands along the gel.
Southern blot hybridization:
The specificity of the PCR products are tested by southern hybridization using specific labeled
DNA probe for botulinum neurotoxin gene (BoNT). The amplified and electrophoresed BoNT
DNA fragments are tarnsfered to a nylon membrane and hybridized with a specific labeled probe
and the hybrization is visualized under X-rays film.

General protocol for Southern blot hybridization:

Background:
DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is
designed to locate a particular sequence of DNA within a complex mixture. For example,
Southern Blotting could be used to locate a particular gene within an entire genome. The amount
of DNA needed for this technique is dependent on the size and specific activity of the probe.
Short probes tend to be more specific.
Steps:
1. Digest the DNA with an appropriate restriction enzyme.
2. Run the digest on an agarose gel.
3. Denature the DNA (usually while it is still on the gel). Soak it in 0.5M NaOH to separate
double-stranded DNA into single-stranded DNA. As only ssDNA can transfer. A depurination
step is optional. Fragments greater than 15 kb are hard to transfer to the blotting membrane.
Depurination with HCl takes the purines out and cuts the DNA into smaller fragments.
4. Transfer the denatured DNA to the membrane. Traditionally, a nitrocellulose membrane is
used, although nylon or a positively charged nylon membrane may be used.Transfer is usually
done by capillary action.Capillary action transfer draws the buffer up by capillary action through
the gel an into the membrane, which will bind ssDNA. After the transfer of DNA to the
membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the
membrane.
5. Probe the membrane with labeled ssDNA. This is also known as hybridization. This process
relies on the ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of
complementary strands. Probing is often done with 32P labeled ATP, biotin/streptavidin or a
bioluminescent probe. A prehybridization step is required before hybridization to block non-
specific sites.
6. Visualize radioactively labeled target sequence by autoradiography if a radiolabeled 32P probe
is used. For biotin/streptavidin detection is done by colorimetric methods, and bioluminescent
visualization uses luminesence.
Conclusion:
As botulism is a potentially lethal disease, a rapid diagnosis is essential. Molecular techniques
combined with immunological methods is an effective way for rapid and sensitive detection of
botulinum neurotoxins. The molecular techniques such as PCR helps in the detection of Bot gene
and is helpful in phenotypically grouping different Bot gene toxotypes (A to E). PCR methods
can readily detect the presence of low levels of C. botulinum DNA but do not detect the presence
or absence of the toxin. To counter this, several immunological techniques can be used to detect
the toxin present in the biological samples, techniques like ELISA, immunoblotting are used.
There are some alternative techniques available for detection of Bot toxin for example,
Radioimmunoassay (RIA) test, and Haemagglutination test can be used instead of ELISA. SDS-
PAGE can be used to characterize different types of BoNts by separating them on the basis of
their molecular weight. For genetic typing, Pulse field gel electrophoresis is performed.
References:
1. Detection of Bacterial Toxins in Food.
http://www.rapidmicrobiology.com/test-method/detection-of-bacterial-
toxins-in-food/
2. Overview of ELISA.
https://www.thermofisher.com/us/en/home/life-science/protein-biology/
protein-biology-learning-center/protein-biology-resource-library/pierce-
protein-methods/overview-elisa.html

3. Southern blotting.
https://askabiologist.asu.edu/southern-blotting

4. Laboratory Diagnostics of Botulism; Miia Lindstro¨m* and Hannu

Korkeala.
5. Methods and difficulties in detection of Clostridium botulinum and its
toxins.T. Grenda, E. Kukier, K. Kwiatek.
6. Current Methods for Detecting the Presence of Botulinum Neurotoxins in
Food and Other Biological Samples. Luisa W. Cheng., Kirkwood M. L and
and Larry H. Stanker.
7. Gel electrophoresis.
https://www.sigmaaldrich.com/technical-documents/articles/biology/
nucleic-acid-electrophoresis.html