LAMP and NUCLEIC ACID BASED TECHNIQUES FOR PLANT DISEASE DETECTION.pptx

1. UNIVERSITY OF AGRICULTURAL SCIENCES BENGALURU
College of Agriculture, V.C. FARM, Mandya
Submitted By,
Sachin. R. K
MSc (Ag)
Plant Pathology
V.C.Farm Mandya
PAT 506 Techniques in Detection and Diagnosis of
Plant Diseases(0+2)
TOPIC : NUCLEIC ACID BASED TECHNIQUES

2. NUCLEIC ACID BASED DETECTION
• Nucleic acid-based assays offer a rapid, sensitive, and specific means for
plant pathogen detection.
• They can be used to detect a wide range of plant pathogens, including
viruses, bacteria, and fungi.
• Nucleic acid-based assays offer many advantages over traditional
detection methods, including
I. The ability to detect a pathogen in a wide range of plant tissues,
II. The ability to detect a pathogen in the absence of disease symptoms,
III. The ability to rapidly and accurately identify a pathogen.

3. TYPES OF NUCLEIC ACID BASED TECHNIQUES
1. Based on Non PCR – LAMP
Microarray
2. Based on PCR- Multiplex
Nested
QPCR
Immune capture PCR

4. LAMP (Loop mediated isothermal
amplification)

5. LAMP stands for Loop- mediated isothermal Amplification.
This technology was developed by Notomi et al.(2000)
The LAMP reaction proceeds at a constant temperature using a strand
displacement reaction.
It is a very sensitive, easy and time efficient method.

6. This may be of use in future as a low cost alternative to
detect certain diseases. It may be combined with a reverse
transcription Step to allow the detection of RNA.

7. Technique
LAMP is an isothermal nucleic acid amplification technique.
In contrast to the polymerase chain reaction(PCR) technology, isothermal
amplification is Carried at a constant temperature, and does not require
a thermal cycler.
It is characterized by the use of the 4 different primers specifically
designed to recognize 6 distinct regions On the target gene

8. Amplification and reaction of gene can be completed in a single
step, by incubating the mixture of sample primers, DNA
polymerase with strand displacement activity and substrate at
constant temperature(about 650
C)

9. Types of Primers used in LAMP
• LAMP is characterized by the use of 4 different primers specifically designed to
recognize 6 distinct regions of the target gene. The four primers used are as follows:
• 1. Forward Inner Primer (FIP): The FIP consists of a F2 region at the 3'end and a
F1c region at the 5'end. The F2 region is complementary to the F2c region of the
template sequence. The F1c region is identical to the F1c region of the template
sequence.
• 2. Forward Outer Primer (FOP): The FOP (also called F3 Primer) consists of a F3
region which is complementary to the F3c region of the template sequence. This
primer is shorter in length and lower in concentration than FIP.

10. • 3. Backward Inner Primer (BIP): The BIP consists of a B2 region at
the 3'end and a B1c region at the 5'end. The B2 region is
complementary to the B2c region of the template sequence. The B1c
region is identical to the B1c region of the template sequence.
• 4. Backward Outer Primer (BOP): The BOP (also called B3 Primer)
consists of a B3 region which is complementary to the B3c region of
the template sequence.

11. • 1. F2 region of FIP hybridizes to F2c region of the target DNA and
initiates complementary strand synthesis using the DNA polymerase
with strand displacement activity, displacing and releasing a single
stranded DNA.

12. • 2. Outer primer F3 hybridizes to the F3c region of the target DNA and
extends, displacing the FIP linked complementary strand. This
displaced strand forms a loop at the 5' end.

13. • 3. This single stranded DNA with a loop at the 5' end serves as a
template for BIP. B2 hybridizes to B2c region of the template DNA.
DNA synthesis is now initiated leading to the formation of a
complementary strand and opening of the 5' end loop.

14. • 4. Now, the outer primer B3 hybridizes to B3c region of the target
DNA and extends, displacing the BIP linked complementary strand.
This results in the formation of a dumbbell shaped DNA.

15. • 5. The nucleotides are added to the 3' end of F1 by DNA polymerase,
which extends and opens up the loop at the 5' end. The dumbbell
shaped DNA now gets converted to a stem loop structure. This
structure serves as an initiator for LAMP cycling, which is the second
stage of the LAMP reaction.

16. • 6. To initiate LAMP cycling, the FIP hybridizes to the loop of the
stem-loop DNA structure. Strand synthesis is initiated here. As the FIP
hybridizes to the loop, the F1 strand is displaced and forms a new loop
at the 3' end.
F2 region at the 3'end and a F1c region at the 5'end.

17. • 7. Now nucleotides are added to the 3' end of B1. The extension takes
place displacing the FIP strand. This displaced strand again forms a
dumbbell shaped DNA. Subsequent self-primed strand displacement
DNA synthesis yields one complementary structure of the original
stem loop DNA and one gap repaired stem loop DNA.

18. • 8. Both these products then serve as template for a BIP primed strand
displacement reaction in the subsequent cycles. Thus, a LAMP target
sequence is amplified 13 fold every half cycle.

20. • The final products obtained are a mixture of stem loop DNA with
various stem lengths and various cauliflower like structures (stem-loop)
DNAs with multiple loops. The structures are formed by annealing
between alternatively inverted repeats of the target sequence in the
same strand.

21. Advantages
• 1. Amplification of DNA takes place at an isothermal condition (63 to 65°C) with greater efficiency.
• 2. Thermal denaturation of double stranded DNA is not required.
• 3. LAMP helps in specific amplification as it designs 4 primers to recognize 6 distinct regions on the
target gene.
• 4. LAMP is cost effective as it does not require special reagents or sophisticated equipment.
• 5. This technology can be used for the amplification of RNA templates in presence of reverse
transcriptase.
• 6. LAMP assay takes less time for amplification and detection

22. Applications
1. LAMP is used in rapid diagnosis of viral, bacterial and parasitic diseases.
2. It helps in the identification of genus and species-specific parasites.
Disadvantages
• Complicated primer design
• To sensitive and prone to contamination and small changes
in condition

23. MICRO ARRAY
A set of DNA sequences
representing the entire set of genes
of an organism, arranged in a grid
pattern for use in genetic testing.

24. • Microarray consists of a solid surface to which biological molecules
are arranged in a regular pattern.
• Applicable in the fields of DNA, proteins, peptides and small
molecules like metabolites and drugs.

25. • Orderly arrangement of thousands of
identified sequenced genes printed on an
impermeable solid support, usually
glass, silicon chips or nylon membrane.
• Thousands of spots each representing a
single gene and collectively the entire
genome of an organism.
• Measurement of Gene Expression.

26. • PRINCIPLE
Hybridization between two DNA strands
Microarrays use relative quantitation : Intensity of a feature is compared
to the intensity of the same feature under a different condition, and the
identity of the feature is known by its position.

27. A DNA microarray is a collection of synthetic DNA probes
attached to designated location, or spot, on a solid surface. The
resulting "grid" of probes can hybridize to complementary
"target" sequences derived from experimental samples to
determine the expression level of specific mRNAs in a sample.

28. The reaction procedure of DNA microarray takes
places in several steps:
1.Collection of samples
• The sample may be a cell/tissue of the organism that we wish to conduct the study on.
•Two types of samples are collected: healthy cells and infected cells, for comparison and
to obtain the results
2.Isolation of mRNA
•RNA is extracted from the sample using a column or solvent like phenol-chloroform.
•From the extracted RNA, mRNA is separated leaving behind rRNA and tRNA.
•As mRNA has a poly-A tail, column beads with poly-T-tails are used to bind mRNA.
•After the extraction, the column is rinsed with buffer to isolate mRNA from the beads.

29. 3. Creation of labeled cDNA
• To create cDNA (complementary DNA strand), reverse transcription of
the mRNA is done.
•Both the samples are then incorporated with different fluorescent dyes for
producing fluorescent cDNA strands. This helps in distinguishing the
sample category of the cDNAs.
4. Hybridization
•The labeled cDNAs from both the samples are placed in the DNA
microarray so that each cDNA gets hybridized to its complementary
strand; they are also thoroughly washed to remove unbounded sequences.

30. 5. Collection and analysis
•The collection of data is done by using a microarray scanner.
•This scanner consists of a laser, a computer, and a camera. The laser
excites fluorescence of the cDNA, generating signals.
•When the laser scans the array, the camera records the images
produced.
•Then the computer stores the data and provides the results
immediately. The data thus produced are then analyzed.
•The difference in the intensity of the colors for each spot determines
the character of the gene in that particular spot.

32. How does a DNA microarray work
• To determine whether an individual possesses a mutation for a particular disease, a scientist first
obtains a sample of DNA from the Diseased sample as well as a control sample - one that does not
contain a mutation in the gene of interest.
• The researcher then denatures the DNA in the samples - a process that separates the two
complementary strands of DNA into single-stranded molecules. The next step is to cut the long
strands of DNA into smaller, more manageable fragments and then to label each fragment by
attaching a fluorescent dye (there are other ways to do this, but this is one common method). The
individual's DNA is labeled with green dye and the control - or normal - DNA is labeled with
red dye. Both sets of labeled DNA are then inserted into the chip and allowed to hybridize - or
bind - to the synthetic DNA on the chip.
• If the Plant sample does not have a mutation for the gene, both the red and green samples
will bind to the sequences on the chip that represent the sequence without the mutation (the
"normal" sequence).
• If the Sample does possess a mutation, the diseased sample DNA will not bind properly to the
DNA sequences on the chip that represent the "normal" sequence but instead will bind to the
sequence on the chip that represents the mutated DNA

33. It has applications in many fields such as:
• Discovery of drugs
• Diagnostics and genetic engineering
• Alternative splicing detection
• Proteomics
• Functional genomics
• DNA sequencing
• Gene expression profiling
• Toxicological research (Toxicogenomics)