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Aplikasi Teknik Molekuler
untuk Deteksi & Identifikasi
Patogen Tumbuhan/Bakteri
Oleh Irda Safni
General approach for bacterial
identification
Different
identification
techniques
Physical methods
Genetical methods
 Based on the
 Based on the
characterization of
characterization of
proteome of the bacteria specifics genes of the
bacteria
Biochemical methods
 Based on the
characterization of
metabolic pathways of
the bacteria
To identify unknown bacteria , results are compared
to databases
Struktur Sel Bakteri
Metabolism of the bacteria
Bacteria are living cells who :
- consume nutrients (carbohydrates, proteins…)
- reject metabolic waste.
Bacteria
Enzymes
Nutrients
Metabolic
waste
Biochemical techniques for identifying
bacteria are based on the
characterization of enzymes and
metabolic waste
Application to the identification:
Physical / morpological method
Gram stain
Gram Positive
Gram Negative
Dinding Sel Gram Negatif &
Gram Positif
Application to the identification :
Biochemical Methods
Example : research the ability to use glucose
Bacteria
inoculation
Yellow color :
Acid pH
incubation
Production of acidic waste by
bacteria
Proves the presence of
enzymes which allow the use
of glucose as nutrient by
bacteria
Medium + glucose + pH indicator :
-green color for pH = 7
-yellow color for acid pH (pH <7)
Bacteria « glucose + »
The API system
The most used API system…
API Strep
 Identification of
Streptococcus species
API Staph
 Identification of
Staphylococcus species
API 20NE
 Identification of Non
Enterobacteria (Pseudomonas
for example)
API 20E
 Identification of
Enterobacteria
The API system
API 20 E after incubation…Positive results for all tests :
API 20 E after incubation…Negative results for all tests :
The API system
Example of results for bacteria to test :
3-Expression of results by software :
Name of the
identified
bacteria
Quality
identification
In this example we have a
very good identification of
bacteria
Salmonella spp
Application to the identification :
Biochemical Methods
Enzyme-Linked Immunosorbent Assay (ELISA):
immunological detection
is a test that uses antibodies and color change to identify a substance.
A. Bind sample to the support (commonly plastic or a membrane)
B. Add primary antibody; wash
C. Add secondary antibody-enzyme conjugate; wash
D. Add substrate
enzyme linked
secondary antibody
E
Y
Y
E
bound primary
antibody
antigenic site
Y
Y
E
iiiiiiiiiiiiiii
Support
E
Y
Y
Target molecule
Y
Y
colorless substrate
colored product
Molecular Methods Used For Detection
of Plant Pathogens
• Polymerase Chain Reaction
• Molecular hybridization
• Molecular markers
• Nucleic acid sequence/Probes
• Micro-arrays
Polymerase Chain Reaction
 PCR is an in vitro method of nucleic acid
synthesis by which a particular segment of
DNA can be specifically replicated.
 Invented by Karry Mullis(1987)
 PCR is an ingenious new tool for molecular
biology for identification of plant pathogens.
PCR Principle:
The double stranded DNA of interest is denatured to
separate into two individual strands each strand is then allowed
to hybridize with a primer (renaturation). The primer template
duplex is used for DNA synthesis (the enzyme DNA
polymerase). These three steps denaturation, renaturation and
synthesis are repeated again and again to generate multiple
forms of target DNA.
Steps in PCR:
I.
Denaturation of DNA:
 Primers will only attach to end subsequently elongate the
Developing nucleic acid chain on a template of single
stranded (ss) DNA
 Thus if the target sequences is double stranded (ds)DNA,
the strand need to be split apart from each other to
produce ssDNA. This is performed by heating the material
to 90-96◦C a process termed as denaturation.
 The step is of 4 min in the first cycle of PCR but of only 2
min in subsequent cycles.
II. Annealing of primers:
 The primers are then attached to the ends of the
segment to be amplified this process is called
annealing.
 This takes place at a temperature range of 3750◦C.
III. Polymerization:
 After the primers are attached they “kick-start
the polymerization which then elongates the
developing nucleic acid chain between them.
 Bases are successfully added on to the
developing new nucleic acid chain according to
the sequence of the segment, which is being
detected, producing a new chain consisting of a
complementary sequence to that of the target
segment.
IV. Amplification:
The new ds DNA need to be split apart again to yield two ss
DNA strands by reheating the mixture to 95˚C i.e. denaturation,
and the cycle is repeated.
 Each cycle resulting in a logarithmic increase in the amount of
DNA which is amplified.
Thus, within 20 cycles a million fold amplification of the starting
amount of nucleic acid can be achieved.
The cyclical variation of temperature of the reaction is carried
out by an automated thermal cycler.
V. Detection of target DNA sequence:
• Amplification
of
DNA
can
be
readily
demonstrated by conventional DNA detection
techniques such as nucleic acid hybridization
or by agarose gel electrophoresis.
Advantages:
 The major advantage of PCR is sensitivity (one
molecule of nucleic acid within 1,00,000 cells).
 It can detect infections at an early stage.
 Useful in detection of non-replicating virus.
 PCR tests are more rapid (result within 24-48
hours).
Disadvantages:
 False positive results due to contamination from
operator, residual matter in testing utensils or
air contamination can result in false positive
reaction.
 Reagents used are still very expensive.
 Useful only for those pathogens for which
primers have been specifically designed.
• PCR and other amplification methods are
extremely sensitive and very specific. For
accurate test interpretation, use proper
controls.
– Positive control: positive template
– Negative control: negative template
– Amplification control: constantly encounter
template unrelated to target
– Reagent blank: no template present
Application of PCR technique in plant pathology:
 Diagnosis and quantification of diseases
 Study of pathogen mating type
 Production of virus free material.
 Field surveys to assess the incidence and
geographic distribution of plant pathogens
 Domestic and international plant quarantine
programmes.
 Detection of mixed virus infections
 Analysis of virus distribution in different
plant tissues.
 Identification of alternative host plant
 Germplasm screening programmes.
 Evaluation of levels of resistance of cultivars.
 Determination of virus
Techniques: RT-PCR
 “Gold standard molecular method” for detection of viruses.
 An RT-PCR (Reverse transcriptase-polymerase chain reaction)
is a highly sensitive technique for the detection and
quantitation of mRNA (messenger RNA).
 The technique consists of two parts:
1) The synthesis of cDNA (complementary DNA) from
RNA by reverse transcription (RT)
2) The amplification of a specific cDNA by PCR.
Schematic diagram of RT-PCR procedure
Many viroids like Apple dimple fruit viroid (ADFVd),Pear
blister canker viroid (PBCVd), Peach latent mosaic viroid
(PLMVd) have been detected by this method (Shamloul et
al.,2002).
It also applied in detection of viruses like CLRV, ACLSV,
Apple
mosaic
virus
and
TomRV.
The sensitivity afforded tends to be similar to ELISA or
hybridization
techniques
(Olmos
et
al.,
2005)
Bio-PCR

BIO-PCR involves an enrichment technique prior
to extraction and amplification of DNA of the
target bacterium (Schaad et al.,1995).
 The intent of the method is to achieve target
bacterium and suppress the growth of non target
bacterium.
 Normally the success of the method is wholly
dependent on the availability of a reliable selective
medium.
Application:
• Detection of Pseudomonas savastanoi pv.
savastanoi by PCR was enhanced by
preenrichment in either non selective King’s B
medium or on a semi-selective medium design
for Pseudomonas savastanoi pv. savastanoi by
PCR (Penyalver et al;2000).
• Song et al. (2004) describe the development of
nutrient enrichment medium for Acidovorax
avenae subsp. avenae on rice seeds.
Advantages:
• BIO-PCR increases the sensitivity of detection.
• It avoids the possibility of detecting dead bacterial
cells.
Limitations:
• Quantification of bacterial population cannot be
readily done.
• If selective medium is lacking sensitivity of
detection is lacking.
Nested PCR
 Sensitivity and specificity problems associated with
conventional PCR and RT-PCR can be reduced by using
nested PCR-based methods.
 It involves the introduction of a second round of
amplication using the amplicon of the first PCR reaction
as template for the second.
 Some authors proposed single-tube nested-PCR protocols
for the bacteria E. amylovora (Llop et al., 2000), for
Pseudomonas savastanoi pv. savastanoi (Bertolini et al.,
2003), and some viruses (Yourno, 1992).
 Bertolini et al., (2003) developed a nested PCR test for
Pseudomonas savastanoi pv. savastanoi using external
primers that amplified at 620C and internal primers that
amplified at 500C.
 A simple device based on the use of a compartmentalized
Eppendorf tube, which enables RT reaction and nested
PCR to be carried out in a single tube and in onemanipulation, has also been described for detection of
Citrus tristeza virus (CTV) (Olmos et al., 1999 and 2003).
 Coupling nested-PCR variants with squashed or printed
samples on paper membranes has allowed the detection
of RNA targets from several viruses in plant material and
in individual insect vectors (Cambra et al., 2006a; Moreno
et al., 2007).
Advantages:
• Sensitivity is increased by two orders of
magnitude reaching about 102 bacterial cells/ml of
extract.
Limitations:
• Need to accurately establish the ratio between
external and internal primers
• Use of limiting amounts of external primers to
avoid interference during the second
amplification.
Multiplex PCR
 This methodology has demonstrated to be a
valuable tool for detection and identification
purposes (Lopez et al, 2006)
 The simultaneous detection of two or more DNA
or/and RNA targets can be afforded by duplex or
multiplex PCR in a single reaction with several
specific primers
 In this case the choice of primers for amplification
depends upon the desired type or detection
Two successful examples are the simultaneous detection of
the six major characterized viruses affecting olive trees:CMV,
Arabis mosaic virus (ArMV), Olive latent virus-1 and
Olive
latent
virus-2
(Bertolini
et
al.,2001)
Nine grapevine viruses (ArMV, grapevine fanleaf virus,
grapevine virus A, grapevine virus B, rupestris stem pittingassociated virus, grapevine fleck virus, grapevine leafrollassociated virus-1, -2 and -3)(Gambino andGrinbaudo,2006).
Scheematic representation of multipex PCR
Contoh Aplikasi Multiplex PCR untuk Deteksi
Bakteri Ralstonia solanacearum species complex
Real Time PCR
 An advancement of m-PCR is the development of Real
Time PCR.
 Real Time PCR is a quantitative procedure, which helps to
detect, accumulation of PCR products during the PCR
reaction.
 PCR products can be monitored using either fluorescent
DNA intercalating dyes such as SYBR Green I, or sequence
specific probe based assays using TaqMan probes.
Taqman R
ABI prism 7900 continuously measures PCR product accumulation using Taqman probe
Sequence of probe is homologous to an internal target sequence
Probe intact
Energy transfer between fluoroaphore
Fluorescent emission is quenched
Probe cleaved by 5’ nuclease activity of Taq polymerase
(extension phase)
Increase in emission intensity is measured
Real Time PCR
• For detection of Clavibacter michiganensis subsp. sepedonicus in
potato tubers (Van Beckhoven et al., 2002),
•
• Ralstonia solanacearum (Weller et al., 2000; Ozakman and Schaad,
2003),
• Erwinia amylovora, the causal agent of fire blight. (Salm and Geider,
2004),
• Candidatus Liberibacter asiaticum (Li et al., 2006),
•
Xanthomonas fastidiosa (Bextine et al., 2005),
• Xanthomonas fragariae (Weller et al., 2007)
Advantages:
 It allows the monitoring of reaction while it is in
course, thus avoiding the risk of contamination.
 Rapid on-side diagnosis of quarantine pathogen.
 It offers faster and more accurate detection
assays.
 Require fewer reagent and allows additional
studies to be performed during detection.
Nucleic Acid Hybridization
 The Basic Process of Binding a Single Strand of
Nucleic
Acid
(DNA
or
RNA)
to
Its
Complementary Strand Is Called Nucleic Acid
Hybridization.
 First utilized in plant pathology to detect Potato
spindle tuber viroid (Owens and Diener, 1981)
Principle:
• Denaturation of the ds DNA can be achieved by
exposure to high temperature or alkaline pH.
Dissociated strands of DNA can be immobilized on a
solid phase support, such as latex, magnetic beads,
microtitre plates ,nitrocellulose or nylon based
membrane, and then hybridized with single strand,
labeled nucleic acid (usually DNA) probe.
• The probes will hybridized only with the denatured
strand of complementary nucleic acid.
DNA-DNA hybridization
Strain 1
Heat
+
0% Homology
Strain 2
100% Homology
a) Dot-Blot Hybridization Assay
 Samples of denatured nucleic acid from healthy and
infected plants are directly spotted on to a prewetted,
solid matrics such as a nitrocellulose or nylon based
membrane.
 Nucleic acids are then firmly immolised on a solid
support by baking the membrane in an oven for 2hr. at
80◦C.
 To block the remaining Free DNA binding sites, the
membrane is incubated several minutes in a sealed
plastic bag with prehybridisation solution.
Continued….
 After removing the prehybridisation solution, the
hybridization solution containing the denatured specific
DNA probe is added to the plastic bag containing the
sample membrane, and incubate for several hours to
allow hybridization between the probe and the target
nucleic acid.
 The membrane is washed several times to remove the
unbound DNA probe following hybridization.
 Hybridization between the target nucleic acid
immobilized on the membrane and the DNA probe is
detected either by autoradiography on X-ray film, or by a
calorimetric reaction.
Continued….
 The sensitivity of dot-blot hybridization may equal or
exceed that of ELISA (Polston et al., 1989).
 This technique has been employed for Apple mosaic
virus(ApMV), Prunus necrotic ringspot virus (PNRSV),
Prune dwarf virus (PDV), and Apple chlorotic leaf spot
virus (ACLSV) (Pallás et al., 1998).
Nucleic Acid Hybridization
Probe present
No probe
b) Fluorescence in situ hybridization
 (FISH) combines microscopical observation of bacteria
and the specificity of hybridization (Volkhard et al., 2000).
 It is dependent on the hybridisation of DNA probes to
species-specific regions of bacterial ribosomes.
 There is a high affinity and selectivity of DNAprobes
because FISH takes place under very stringent
hybridisation conditions.
 In practice, FISH can reach a relatively low sensitivity
levels in some cases, even though has been employed in
some recent works (Ercolini et al.2006).
3. Molecular markers:
a) Restriction fragment length polymorphisms:

This technique makes use of restriction enzymes to fragment DNA,
which is then separated by agarose gel electrophoresis.

Each restriction enzyme recognizes a specific nucleotide sequence
and cuts the DNA specifically every time the sequence occurs.

The fragmented DNA is separated by gel electrophoresis, and the
banding pattern is made visible by staining (ethidium bromide) or
by autoradiography.

When mitochondrial DNA (mtDNA) from different species are
examined, differences in the size and number of restriction
fragments can be detected.

These differences can result from inserts or deletions between
existing restriction-enzyme sites or mutations that destroy or
create restriction sites.
b) RAPD Markers:
 RAPDs are DNA fragments amplified by the PCR using short
synthetic primers of random sequence.

These oligonucleotides serve as both forward and reverse primer,
and are usually able to amplify fragments from 1-10 genomic sites
simultaneously.
 Amplified fragments are separated by agarose gel electrophoresis,
and polymorphism are detected.

These polymorphisms are considered to be primarily due to
variation in the primer annealing sites, but they can also be
generated by length differences in the amplified sequence between
primer annealing sites.
Some of specific DNA fragments detected in a
profile may be cut out of the gel and sequenced
to obtain a SCAR (Sequence-characterized amplified region), into which specific primers can be
designed for a more precise PCR detection.
SCAR primers have been used to specifically identify Phytophthora
cactorum (Causin et al., 2005), Fusarium subglutinans (Zaccaro et
al., 2007) and Guignardia citricarpa (Stringari et al., 2009)
RAPD also useful for the analysis of the genetic diversity among
populations.
Ex. Fusarium spp. ( Arici & Koc 2010) and pathotypes of Elsinoë spp.
(Hyun et al., 2009).
Continued….
The RAPD technique is rapid, inexpensive and does
not require any prior knowledge of the DNA sequence of
the target organism.
Results obtained from RAPD profiles are easy to
interpret because they are based on amplification or
non amplification of specific DNA sequences.
RAPD analyses can be carried out on large numbers
of isolates without the need for abundant quantities of
high-quality DNA (Nayaka et al., 2011).
Arbitrarily Primed PCR: Random
Amplification of Polymorphic DNA (RAPD)
MO
M = Molecular weight marker
O = Outbreak strain
Four isolates differ from the outbreak strain.
Other Genotypic Methods Used to
Type Organisms
• Plasmid fingerprinting with restriction
enzymes
• RFLP analysis
• Amplified Fragment Length Polymorphism
(AFLP)
• Interspersed repetitive elements
• Ribotyping
• spa typing
• Multilocus sequence typing
Interspersed Repetitive Elements
REP sequence inverted repeat
….GTGAATCCCCAGGAGCTTACATAAGTAAGTGACTGGGGTGAGCG….
ERIC sequence inverted repeat
PCR amplification priming outward from repetitive elements
generates strain-specific products.
GCC G/T GATGNCG G/A CG C/T NNNNN G/A CG C/T CTTATC C/A GGCCTAC
Isolate A
Isolate B
M
A
B
Is the unknown (U) strain A or B?
M
A
B
U
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
1. 1 kb ladder
2. BP 1001 (BS 3748)
3. BP 1002 (BS 3828)
4. BP 1003 (BS 0258)
5. BP 1004 (BS 0320)
6. BP 1005 (BS 3792)
7. BP 1006 (BS 3793)
8. BP 1007 (Pap017)
9. BP 1024 (Pap042)
10. BP 1056 (BS 0324)
11. BP 1057 (BS 0588)
12. BP 1058 (BS 0651)
13. BP 1059 (BS 0701)
14. BP 1060 (BS 0702)
15. BP 1061 (BS 0708)
16. BP 1062 (BS 0709)
17. BP 1063 (BS 0710)
18. BP 1064 (BS 0711)
19. BP 1065 (BS 0712)
20. BP 1066 (BS 0713)
21. BP 1067 (BS 0714)
22. BP 1068 (BS 0912)
23. BS 0291T (Positive control)
24. Water (negative control)
25. 1 kb ladder
BOX-PCR
4. Nucleic Acid Sequence /probes:
a) Nucleic Acid Sequence Based Amplification (NASBA):
It can be used to detect RNA targets and based on exponential
amplification of single stranded RNA molecules by simultaneous
activities of reverse transcriptase and polymerase enzymes (Kievits
et al., 1991).
The reaction requires the use of three enzymes:
i) Avian myeloblastosis virus reverse transcriptase( AMV-RT) for
reverse transcription and to obtain double stranded cDNA,
ii) RNase H to hydrolyze the RNA fragment of the hybrid molecule
DNA- RNA
iii) T7 RNA polymerase to produce a large amount of anti-sense,
single strand RNA transcripts corresponding to the original RNA
target
Continued….
 This technology has been applied for detecting plant viruses such as
Apple stem pitting virus (Klerks et al., 2001)
 PPV (Olmos et al., 2007a)
 Potato virus Y, ArMV and the bacteria Clavibacter michiganensis
subsp. sepedonicus and Ralstonia solanacearum (Szemes and
Schoen, 2003).
 The sensitivity of this method has proven similar to that obtained by
real-time RT-PCR when applied to PPV detection (Olmos et al.,
2007a).
b) Loop-mediated isothermal amplification (LAMP):
 The method requires a set of four specifically designed primers that
recognize six distinct sequences of the target and a DNA polymerase
with strand displacement activity.
 The amplification products are stem-loop DNA structures with
several inverted repeats of the target and cauliflower-like structures
with multiple loops.
 The amplification takes place at 60-65ºC for 60 min.
 Although it was initially developed for DNA it can be adapted to
amplify RNA (RT-LAMP) (Fukuta et al., 2003).
 The method has only been applied to the detection of some plant
viruses such as PPV, with a sensitivity level similar to that obtained
by real-time PCR (Varga and James, 2006).
5. Microarray technology:
 Microarrays are generally composed of thousands of
specific probes spotted onto a solid surface (usually
nylon or glass).
 Each probe is complementary to a specific DNA
sequence (genes, ribosomal DNA) and hybridisation with
the labeled complementary sequence provides a signal
that can be detected and analysed.
 Until now, the microarray technology focuses its use in
multiplex format of similar or very different pathogens,
taking advantage of the number of probes that can be
employed in one chip (Pasquini et al., 2008).
continued…
 Another type of microarray under development
is called the nanochip (Sosnowski et al., 1997)
 Based on an electronically addressable
electrode array that provides direct electric field
control over the transport of charged molecules
to selected micro locations and concentration
over an immobilized substrate
Future Perspectives:
 Information resulting from detection by improved molecular methods could be
used to optimize diseasecontrol through more rational decisions about the
choice and use of control measures.
 Besides optimization of PCR and real-time PCR protocols, the advances in
microarray, microchip or biochip technology will allow to test simultaneously,
the prospect of a wide variety of pathogenic microorganisms, and the potential
of this tool will open new fields of studies in plant pathology.
 Since cultivated plants can be affected by diseases caused by many types of
organismsa method able to detect several pathogens simultaneously would be
ideal for testing plant material, especially for quarantine pathogens.
 Protocols based on PCR have already been developed for the most important
pathogens and they should be optimized soon, looking for multiplex detection,
trying to simplify the RNA or DNA extraction without decreasing the
robustness of the methods
Conclusion:
 Nucleic acid-based methods are sensitive, specific and allow genetic
relationships to be determined.
 In plant pathology compared to traditional methods, PCR offers
several advantages, because, organisms do not need to be cultured
before their detection.
 It affords high sensitivity at least theoretically, enabling a single
target molecule to be detected in a complex mixture.
 It is also rapid and versatile.
 In fact, the different variants of PCR, have increased the accuracy of
detection and diagnosis, and opened new insights into our
knowledge of the ecology and population dynamics of many
pathogens.
 Providing a valuable tool for basic and applied studies in plant
pathology.