Lecture 1

Introduction to
molecular diagnostic
techniques in
agriculture, health,
environment and food
 Effectivesurveillance of pathogens can
be achieved through a combination of
the conventional and molecular
techniques
 The conventional or cultural standard
methods appear to have been used since
the inception of microbiological sampling
 These methods mainly involve plating
onto selective agar and confirmation of
presumptive pathogen colonies by
biochemical tests
 Theyare widely used and have the
advantage that
 they are cheaper,
 detect only viable pathogens
 yieldisolates that can further be
characterized and studied
 However, they are
 Laborious
 relatively slow
 less efficient
 Molecular techniques have also been widely
used
 in surveillance,
 mutation and other genetic studies of
pathogens to increase our understanding into
the primary source of pathogens
 source of infection
 genetic diversity.
 Molecular techniques have the advantage
that, as compared to the conventional
method, they are:
 rapid,
 less laborious,
 more sensitive
 specific
 efficient
 The application of molecular
techniques for detecting and typing of
pathogens in surveillance studies
provide reliable epidemiological data
for tracing the source of infections.
 A wide range of molecular techniques
have been used for detecting,
speciating, typing, classifying and/or
characterizing pathogens of great
significance to humans.
Molecular Diagnostic Techniques
 Detection methods using polymerase chain
reaction (PCR)-based assays
 Single PCR
 Multiplex PCR
 Real-time PCR (qPCR)
 RT-PCR
Molecular Diagnostic Techniques
 Molecular techniques for typing pathogens
 Pulsed field gel electrophoresis (PFGE)
 Multilocus sequence typing (MLST)
 Random amplified polymorphism
deoxyribonucleic acid (RAPD)
 Amplified fragment length polymorphism
(AFLP)
 Restriction fragment length polymorphism
(RFLP)
 Plasmid profile analysis
 DNA sequencing
 Other techniques
 Detection methods using polymerase
chain reaction (PCR)-based assays
 PCR is an in situ DNA replication process that
allows for the exponential amplification of
target DNA in the presence of synthetic
oligonucleotide primers and a thermostable
DNA polymerase
 A wide range of different concentrations or
 units of DNA templates (5–25 ng)
 Taq DNA polymerase (0.6–1.25 U)
 primers (0.11–10 μM)
 temperature cycles (45–95.8 °C and 30–40 cycles)
have been employed to detect or confirm pathogens
 Detection methods using polymerase
chain reaction (PCR)-based assays
 Other components of a PCR reaction such as
 deoxyribonucleotide triphosphates (dNTPs),
 magnesium (Mg2+)
 buffersolutions have been used in different
concentrations to increase detection limits.
 Detection methods using polymerase
chain reaction (PCR)-based assays
 A PCR process may involve the use of
 one primer (single PCR)
 multiple primers (multiplex PCR)
to detect pathogen isolates.
 Other forms of PCR are:
 real-time PCR (qPCR)
 reverse-transcription PCR (RT-PCR)
 many more…..
Single polymerase chain reaction
 PCR involving the use of a single primer
set (which targets a specific gene) to
detect an organism.
 The primer set can be designed for
specific species and can detect the target
organism in the presence of others.
 This kind of PCR can be applied to rapidly
detect or identify pathogen directly from a
sample (food, water, clinical or
environmental) with or without pre-
enrichment.
Single polymerase chain reaction
 However, direct detection of pathogens by
PCR assays in the environment or in sample
of turbid nature can result in the detection of
DNA in dead cells and give false negative
results
 Enrichment before PCR detection and/or the
application of fluorescence in situ
hybridization (FISH) techniques have been
suggested to curb this situation
Single polymerase chain reaction
 PCR using universal or specific primers
have been used to initially amplify the 16S
rRNA genes of pathogen before being
sequenced to help in the identification of
unknown or novel pathogen species.
Multiplex polymerase chain reaction

 This is a modification of polymerase chain

reaction that uses multiple primers within a
single PCR mixture to detect, identify
and/or differentiate pathogens.
 Thus, in multiplex PCR more than one
target sequences are amplified in a reaction
to produce amplicons of varying sizes
specific for different DNA sequences.
Multiplex polymerase chain reaction
 Multiplex PCR
 reduces cost
 limits volume of samples
 allows for the rapid detection of multiple
pathogen species, strains and so on
 primer design is critical in the
development of multiplex PCR
 All primers need to have close annealing
temperature, the amplicons must be
markedly different in sizes
 multiple primers may interfere with each
other during the amplification process
 Real-time PCR (qPCR)
 Real-time PCR is a polymerase chain reaction
process in which the target DNA is amplified
and quantified simultaneously within a
reaction.
 Real-time PCR employs specific primer set,
one or two probes and/or fluorescent dye to
improve detection signals
 In real-time PCR, the amplified DNA is
detected in real time as the reaction progresses
instead of at the reaction end.
 Real-time PCR shortens detection time
compared to standard PCR and can determine
the absolute or relative number of pathogens in
various samples
 Real-time PCR (qPCR)
 Thereis no post-PCR processing of
products, leads to high throughput and
reduces the risk of amplicon contamination
by laboratory environments
 However, equipment and reagent costs are
high for real-time PCR
Real-treverse-transcription PCR (RT-PCR)

 In reverse-transcription PCR, RNA is used

as the initial template instead of DNA.
 Reverse transcriptase is used to reverse
transcribed the target RNA into its DNA
complement (cDNA) and amplified using
PCR
 Reverse-transcription PCR is useful in
detecting only viable cells of pathogens;
 However RNA is unstable requiring much
skill during handling and quantification for
pathogen detection
Pulsed field gel electrophoresis (PFGE)
 PFGE is an agarose gel electrophoresis
technique used for separating larger pieces
of DNA by applying electrical current that
periodically changes direction (three
directions) in a gel matrix unlike the
conventional gel electrophoresis where the
current flows only in one direction
 In PFGE, intact chromosomes are digested
using restriction enzymes or restriction
endonucleases to generate series of DNA
fragments of different sizes (also known as
restriction fragments length polymorphisms,
RFLPs) and patterns specific for a particular
species or strain
Pulsed field gel electrophoresis (PFGE)

 Advantages
 Has high discriminatory power,
reproducibility and typeability
 Disadvantages
 Requires 3–5 days to complete a test, the
cost is relatively high compared to other
methods,
 This technique has limited availability
 References: Wassenaar and Newell (2000); Trindade et al. (2003)
Multi-Locus Sequence Typing (MLST)

 MLST is a an unambiguous, portable and

nucleotide-based technique for typing
pathogens using the sequences of internal
fragments of (usually seven) house-
keeping genes
 In MLST, different sequences within a
pathogen species are assigned as distinct
alleles for each house-keeping gene and the
alleles at each end of the seven loci define
the allelic profile or sequence type for each
isolate
Multilocus sequence typing (MLST)

 Approximately 450–500 bp internal

fragments of each gene are used and most
pathogens have enough variation within the
house-keeping genes to provide many
alleles per locus thus allowing billions of
distinct allelic profiles to be differentiated
utilizing the seven house-keeping loci
Multilocus sequence typing (MLST)
 Advantages
 It provides typing data that are
 unambiguous,
 portable,
 more accurate
 more discriminatory for most bacteria
 These data are readily available, comparable and
accessible via the internet in contrast to most typing
procedures involving comparison of DNA fragment
sizes on a gel
 This makes MLST typing data more suitable for
global epidemiological studies.
 Disadvantages
 It is expensive compared to RAPD, ERIC, REP and
plasmid analysis
 References: Wassenaar and Newell (2000); Trindade et al. (2003)
 Random amplified polymorphism
deoxyribonucleic acid (RAPD)
 RAPD is a PCR-based technique in which
arbitrary primers (typically 10-mer primers)
are used to randomly amplify segments of
target DNA under low-stringency PCR
condition
 This process leads to the amplification of
one or more DNA sequences and generates
a set of finger printing patterns of different
sizes specific to each strain
Random amplified polymorphism
deoxyribonucleic acid (RAPD)

 Advantages
 It is relatively cheap, rapid, readily
available, and easy to perform
 Disadvantages
 RAPD has low reproducibility, average
discriminatory power and approximately
80 % typeability
 Amplified fragment length
polymorphism (AFLP)
 AFLP involves the use of two restriction
enzymes to digest total genome DNA, one
with an average cutting frequency (4-bp
recognition site) and the other with a higher
cutting frequency (6-bp recognition site)
followed by linking of adapters to the sticky
ends of the restriction fragments and
amplification of a subset of selected
restriction fragments
 Amplified fragment length
polymorphism (AFLP)
 The primers used for amplification are
radioactive or fluorescent labelled and
denaturing polyacrylamide gel analysis is
used to determine the presence or absence
of DNA fragments to identify
polymorphisms
 Amplified fragment length
polymorphism (AFLP)
 Advantages
 good discriminatory power
 good reproducibility
 100 % typeability
 needs no prior sequence information for
amplification
 insensitive to genetic instability
 Disadvantages
 complex method
 requires 3–4 days to complete a test
 requires major capital investment
 Restriction fragment length
polymorphism (RFLP)
 Ituses restriction enzyme to digest DNA
and to separate the resulting restriction
fragments according to their length on
agarose gel electrophoresis
 Restrictionfragments are then transferred
into a membrane through Southern blot
procedure and hybridized to a membrane
bound labelled DNA probe
 This method utilises the variations in
homologous DNA sequences to characterize
pathogens
 Restriction fragment length
polymorphism (RFLP)

 Advantages
 Inexpensive
 verysensitive for strain identification or
differentiation
 had widespread application
 Disadvantages
 Thetechnology is also slow, difficult and
could take up to a month to complete
 Plasmid profile analysis
 Plasmid profile analysis is one of the oldest
molecular techniques used for epidemiological
investigation.
 In this technique, plasmid DNAs are extracted
from bacteria and the DNA is separated on
agarose gel electrophoresis.
 It is easy to perform this technique and to
interpret the results except that plasmids are
mobile extrachromosomal elements that can
spontaneously be lost or readily acquired by
bacteria and thus isolates that are related
epidemiologically can easily display different
plasmid profiles
 DNA sequencing techniques
 DNA sequencing techniques involve
technologies used to determine the order of
the nucleotide bases in a DNA molecule
 In recent times, DNA sequencing is widely
and routinely used in the identification,
typing, characterization and/or taxonomic
classification of unknown or novel
pathogens isolates by many researchers.
 DNA sequencing techniques
 DNA sequencing has always been preceded
by PCR to amplify the target genes.
 16S rRNA is a common gene that is
amplified for sequencing and subsequently
for the identification, typing and/or
taxonomic classification of the pathogen in
question.
 DNA sequencing techniques
 Advantages
Sequencing has
 high discriminatory power,
 100 % typeability
 good reproducibility
 Disadvantages
 It requires 2–3 days to complete a test,
 has limited availability
 costs higher than other typing methods
 Other typing methods
 Enterobacterial
Repetitive Intergenic
Consensus (ERIC)
 Repetitive Extragenic Palindromic (REP)
 Ribotyping
 Conclusion
 Severaldetection and typing methods have
been developed and are widely used to
detect,
differentiate,
type
classify
pathogens for
efficient identification,
outbreak investigations,
clinical treatments
epidemiological studies.
 Conclusion
 The combination of two or more primers
and/or methods, and optimization of
methods will drastically increase the
discriminatory power of the detection or
typing technique employed.