PCR & NON PCR BASED MARUTHI.pptx

2. UNIVERSITY OF AGRICULTURAL SCIENCES, BANGLORE
COLLEGE OF AGRICULTURE, MANDYA
MOLECULAR TECHNIQUES FOR DETECTION OF PLANT VIRUSES:
PCR AND NON-PCR BASED
.
SUBMITTED BY:
MARUTHI H PATIL
PAMM2006
I YEAR PH.D

3. INTRODUCTION:
 Plant pathogenic bacteria, phytoplasmas, viruses, and viroids
cause destructive, extensive, and inexpensively important
diseases in a very extensive range of plant species worldwide.
The damage is often sufficient to cause significant yield losses
in cultivated plants.
 The prevention measures demand pathogen detection
methods of high sensitivity, specificity, and reliability because
many phytopathogenic bacteria and viruses can remain latent
in “subclinical infections,” and/or in low numbers, and/or in
some special physiological states in propagative plant material
and in other reservoirs.
 Here, we present the state of the art of different
morphological, bio-chemical, serological, and molecular
detections of plant pathogenic viruses.

4.  Pathogenic detection refers to the presence of a
particular target organism in plant tissues,vectors, plant
products, or environmental samples, with emphasis on
symptomless plants, whereas Diagnosis is related to the
identification of the nature and cause of a disease in
plants showing symptoms.
 Accurate identification and early detection of
pathogensis the cornerstone of disease management in
many crops. Many plant pathogens are difficult to
identify using morphological criteria, which can be time
consuming and challenging and requires extensive
knowledge in taxonomy.
 Molecular detection techniques can generate accurate
results rapidly enough to be useful for disease
management decisions.

5. Molecular Techniques for Detection Of Plant
Pathogen:
 PCR
 Molecular Hybridization
 DNA Microarray Non PCR

6. PCR ( 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.
Principle:
 The double standard 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.

7. Steps in PCR:
1. Denaturation of DNA
Primers will only attach to end subsequently elongated the
developing nucleic acid 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 ss DNA. This is
performed by heating the material to 90 to 96o C a process termed as
denaturation. The step is of 4 minutes in the first cycle of PCR but of
only 2 minutes in subsequent Cycles.
2. 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
temperature range of 37 to 50oC.

8. 3. Polymerization:
After the primers are attached they" kick- start" the polymerization
which then elongates the developing nucleic acid chain between
them. bases are successful 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.
4. Amplification:
The new ds DNA need to be split apart again to yield two ss DNA
strands by reheating the mixture to 95o 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.

11. Advantages:
 It can detect infections at an early stage.PCR tests are more rapid (
result within 24-48 hours).Useful in detection of non- replicating virus.
 Increased ability to detect less common organisms such as viruses.
 PCR has also been used throughout the field of molecular biology,
helping researchers clone and sequence genes for the detection of
mutations.
 PCR is a very sensitive technique that allows rapid amplification of a
specific segment of DNA. PCR makes billions of copies of a specific
DNA fragment or gene, which allows detection and identification of
gene sequences using visual techniques based on size and charge.

12. Disadvantages:
 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.

13. Application of PCR technique in Plant Pathology:
 Diagnosis and quantification of disease
 The use of Polymerase Chain Reaction (PCR) in
infectious disease diagnosis, has resulted in an ability to
diagnose early and treat appropriately diseases due to
fastidious pathogens, determine the antimicrobial
susceptibility of slow growing organisms, and ascertain
the quantum of infection.
 PCR can also help farmers detect the presence of
pathogens that have long latent periods between
infection and symptom development. Moreover, it can
quantify pathogen biomass in host tissue and
environmental samples, and at the same time detect
fungicide resistance.

14. Different types PCR :
1.Multiplex PCR :
 Multiplex PCR is an adaptation of PCR which allows
simultaneous amplification of many sequences. This
technique is used for diagnosis of different diseases in the
same sample.
 Multiplex PCR can detect different pathogens in a single
sample .Also it can be used to identify exonic and intronic
sequences in specific genes and determination of gene
dosage .
 This is achieved when in a single tube include sets of
specific primers for different targets.

16. 2.Nested PCR :
 This PCR increases the sensitivity due to small amounts of the
target are detected by using two sets of primers, involving a
double process of amplification. The first set of primers allows a
first amplification. The product of this PCR is subjected to a
second PCR using the second set of primers.
 These primers used in the second PCR are specific to an internal
amplified sequence in the first PCR. Therefore, specificity of the
first PCR product is verified with the second one.
 The disadvantage of this technique is the probability of
contamination during transfer from the first amplified product
into the tube in which the second amplification will be
performed.

18. 3.Reverse Transcriptase PCR (RT-PCR):
 This PCR was designed to amplify RNA sequences (especially
mRNA) through synthesis of cDNA by reverse transcriptase (RT).
Subsequently, this cDNA is amplified using PCR.
 This type of PCR has been useful for diagnosis of RNA viruses, as
well as for evaluation of antimicrobial therapy. It has also been used
to study gene expression in vitro, due to the obtained cDNA retains
the original RNA sequence.
 The main challenge of using this technique is the sample of mRNA,
because this is considered difficult to handle by low level and
concentration of mRNA of interest and low stability at room
temperature together with sensitivity to action of ribonucleases and
pH change.

20. 4.Real-time PCR:
 It is the technique of
collecting data throughout
the PCR process as it
occurs, thus combining
amplification and detection
into a single step.
 This is achieved using a
variety of different
fluorescent chemistries that
correlate PCR product
concentration to
fluorescence intensity.

21. Molecular Hybridization:
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.
Principle:
Denaturation of the ds DNA can be achieved by exposure to
high temperature or alkaline pH. Dissociation strands of DNA can
be immobilized on a solid phase support, such as latex, magnetic
beads, microtitre plates , nitrocellulose or nylon based member and
then hybridized only with the denatured strand of complementary
nucleic acid.

22. A) Dot- Blot Hybridization Assay:

23. ADVANTAGES
 Dot blot technique does not require the separation of
bands on the solid support medium (agarose), or there
is no requirement of electrophoresis.
 One can detect the presence or absence of genes from
the sample of transgenic individuals in a single test run.
 It does not involve immobilization of the biomolecules
from a gel matrix to the filter membrane.
 Dot blot technique aids in direct blotting of
biomolecule onto the membrane.
DISADVANTAGES
• Dot blot method does not give any qualitative information
about the target biomolecules’ size and molecular weight.
• It does not provide a basis for comparing an original and a
modified target biomolecule within the same slot.

24. B) Fluorescence insuit hybridization:
Fluorescence in situ hybridization is a molecular cytogenetic technique
that uses fluorescent probes that bind to only those parts of a nucleic
acid sequence with a high degree of sequence complementarity.
 FISH combines microscopical observation of bacteria and the
specificity of hybridization.
 It is dependent on the hybridization of DNA probes to species -
specific region of bacterial ribosomes.
 There is a high affinity and selectivity of DNA probes because
FISH takes place under very stringent hybridization conditions.

26. • Advantages: Fluorescent system has two major advantages that make up for
this relatively minor difficulty.
• Observable increase in sensitivity. It is possible to produce preparations
in which the background fluorescence signal is virtually null under optimal
hybridization conditions, thus achieving high signal-to-noise ratios.
• The combination of such high- quality preparations with any of the highly
sophisticated fluorescence microscope systems, such as confocal
microscopes or cool charge-coupled device (CCD) cameras, can detect
accurately in comparison to enzymatic detection methods.
• Enhanced resolution. This is specially useful when more than one target
sequence is being analysed at the same time.
• The fluorescent protocol can be employed for double or triple
hybridization with multiple probes that can easily be detected using the
appropriate non-overlapping fluorochromes.
• In such case, the location of the different target sequences can be
determined without the constraints inherent to transmitted light
microscopy.
• limitations:
• The main limitation of this protocol is the short-lived nature of the
fluorescent signal.
• Unlike the products of enzymatic reactions, fluorescence vanishes away
over time, and bleaches out rapidly when observed under the microscope.
• Therefore, fluorescence-based preparations are temporary.

27. DNA Microarray:
 DNA microarrays or biochips are made of a suface on
which multiple capture probes are linked, each one being
specific for a DNA or RNA sequence of the targets.
 Their purpose is the detection of numerous sequences in a
single assay.Up to 30,000 DNA probes (gene sequences)
can be arrayed onto a single chip.
 DNA microarrays are microscope slides that are printed
with thousands of tiny spots in defined positions, with each
spot containing a known DNA sequence or gene.

28.  complementary sequences will bind to
each other. The unknown DNA
molecules are cut into fragments by
restriction endonucleases; fluorescent
markers are attached to these DNA
fragments. These are then allowed to
react with probes of the DNA chip.

30. CONCLUSION:
 In plant pathology compared to traditional methods , PCR
offers several advantages, because organism 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 v, 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.

31. References:
 Miller, S.A., and Martin, R.R. 1988. Molecular diagnosis of plant disease.
Annu. Rev. Phytopathology. 26: 409–432.
 Fox, R.T.V. (1993). Princtpl.es of diagnostic techntques in plant
Pathology. CAB International, Wallingford UK.
 Mackay IM. 2004. Real-time PCR in the microbiology laboratory. Clin
Microbiol Infect. 10(3):190–212.
 Lévesque, C.A. 1997. Molecular detection tools in integrated disease
management: overcoming current limitations. Phytoparasitica, 25: 3–7.