detection of plant virus using nucleic acid

1. NUCLEIC ACID-
BASED ASSAY FOR
THE DIAGNOSIS OF
VIRAL PATHOGENS.
PRESENTED BY
ABHISEK JENA
A-2023-30-083
SUBMITTED TO
Dr. Shikha Sharma

2. 1 INTRODUCTION
2 IMPORTANCE
3 TYPES OF DETECTION METHOD
4
DNA HYBRIDIZATION
METHODS
5 PCR AND ITS DIFFERENT FORMS
8 MICROARRAY AND RCA
10 CONCLUSION
CONTENT

3. 03
2-Procedure for detection of a plant virus;-
• Determine the plant species
• study the symptoms
• look for the potential virus vector on the plant
• access distribution pattern and incidence of disease
• Go to literature
• compare the fact
• select one or more assays
INTRODUCTION;-
1-What is diagnosis and detection ?

4. Biological assays Molecular assays
• Required indicator plant
• More costly
• Tidicious process
• it requires expensive facilities like
greenhouse,Glass house,screen
house etc.
• Moreover different plant frequently
react to different viruses with the
same symptoms.
• No need of indicator plant
• More specific and Robust
05
WHY TO USE NUCLEIC ACID TECHNIQUES TO
DETECT PLANT VIRUSES?

5. Biological assays
Host range
symptom
method of transmission
Viral nucleic acid assays
Inclusion bodies
Nucliec acid hybridization(PCR,RTPCR)
RCA(Rolling circle amplification)
RPA(Recombinase polymerase amplification)
Microarray
Deep sequencing(Next gen. sequencing)
Viral protien assays Serological assays
04
AVAILABLE DIAGNOSTIC AND/
DETECTION ASSAYS

6. NUCLEIC ACID BASED
TECHNIQUES
• PCR and its variants
• Realtime PCR
• Dot Blot
• Southern Blot
• NOrthern Blot
• Microarray
• Rolling circle amplification
06

7. DOT BLOT HYBRIDIZATION???
07
The dot-blot hybridization is a nucleic acid
hybridization technique where
complementary single-stranded sequences
of the probe (either RNA or DNA) hybridizes
with single-stranded sequences of the test
samples (either RNA or DNA) under suitable
conditions of temperature and salt
concentration.

8. cont...
This hybridization format simply answers the question of whether a plant is or
is not infected by a given virus. Dot blotting does not distinguish between the
number and size of hybridized mole cules since the hybridization signal is the
sum of all sequences recognized by the probe.
However, the technique is rapid and versatile identifying specific nucleic acid
sequences in samples ranging from crude plant sap to highly purified prepara
tions.
08

9. southern blotting
08
SOUTHERN AND
NORTHERN BLOTTING.
Detect DNA
sequences

10. Northern blotting
08
Detect RNA
sequences

11. POLYMERASE
CHAN REACTION
DENATURATION
ANNEALING
EXTENSION

12. • Temperature 94 °C to 98 °C
• Double stranded DNA melt----
Single stranded DNA.
DENATURATION

13. The primers are annealed
(Annealing) with each strand of the
target DNA, when the two strands
of the template DNA are denatured
to become single standed
ANNEALING
AND
EXTENSION
PCR ANNEALING
Then new DNA stand is synthesized
(Chain extension) by adding dNTPs
which are joined one by one
depending upon the complementary
sequence in template DNA by a heat
stable DNA polymerase called Taq
DNA polymerase.

14. This technique is commonly used in molecular
biology to detect RNA
RT-PCR is used to reverse transcribe the RNA of
interest into its complementary DNA (cDNA)
through the use of reverse transcriptase.
Subsequently, the newly synthesized cDNA is
amplified using traditional PCR.
OTHER PCR
FORMAT FOR
DETECTION
Reverse Transcription PCR (RT-
PCR)

16. MULTIPLEX-PCR:
• CONSISTS OF MULTIPLE PRIMER
SETS WITHIN A SINGLE PCR
MIXTURE TO PRODUCE
AMPLICONS OF VARYING SIZES
THAT ARE SPECIFIC TO
DIFFERENT DNA SEQUENCES.
• BY TARGETING MULTIPLE GENES
AT ONCE, ADDITIONAL
INFORMATION MAY BE GAINED
FROM A SINGLE TEST-RUN THAT
OTHERWISE WOULD REQUIRE
SEVERAL TIMES THE REAGENTS
AND MORE TIME TO PERFORM.

17. This technique combines DNA amplification with detection and
quantification of the PCR product in real time in a single tube
Methods based on changes in fluorescence proportional to the
increase in either specific or non-specific PCR product. The
fluorescence is monitored during each PCR cycle to provide an
amplification plot allowing the user to follow the reaction.
In stead of measuring the endpoint focus is on the first
significant increase in the amount of PCR product.
The time required to measure the first significant increase
in PCR product inversely proportional to the initial amount
of DNA template
Principle;-
REAL-TIME
PCR

18. MICROARRAY
Microarray technique is used for the simultaneous detection of multiple
pathogens infecting plants in a single reaction. This method uses virus-
specific oligos immobilized on a membrane or glass slide as probe. The total
RNA isolated from infected plant is converted into cDNA and amplified
through PCR using pathogen-specific primers, labelled using suitable
molecules for detection. The amplified and labelled products are then
applied to the array and allowed for hybridization. After washing, array will
be developed depending on the label used and result visualized using CCD
with suitable software.

19. • Expensive
• Production of large
result at a time
requires more time
to analyse
• provides data for
thousand of genes in
real time
• First and easy to obtain
result
• Promissing to cure the
disease caused by the
viruses
Merits Demerits
MICROARRAY

20. ROLLING CIRCLE
AMPLIFICATION
• This is the only reaction that makes it possible to
synthesize NA products with the desired (artificially
preset) nucleotide sequence.
• A characteristic feature of RCA is the use of circular
DNA templates; the appropriate design of these
templates makes possible a combination with other
enzymatic reactions and various reporter systems, the
performance of bioanalysis in solution, on the surface
of solid materials or in living cells, and the conversion
into high-performance formats.
• In contrast to other amplification methods, RCA can
be used not only as a technique for the amplification
and subsequent detection of the molecules of an
analyte, NA, but also as a tool for obtaining functionally
active NAs that mediate the detection of other
biotargets.

21. LAMP
Loop mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid
amplification method. LAMP exhibits increased sensitivity and specificity due to an
exponential amplification feature that utilises 6 different target sequences simultaneously
identified by separate distinct primers in the same reaction . LAMP assays are significantly
rapid, and do not require expensive reagents or instruments, which aids in cost reduction for
coronavirus detection.

22. BENEFITS ???
• Rapid detection and identification of pathogens for which traditional techniques
are inadequate.
• Detection and identification of patho gens of potential regulatory concern or of
high economic consequences.
• Determining the geographic origin of, or genetic relationships among, pathogen
strains
• Documenting the existence and relative importance of latent infections.

23. LIMITATION ???
• It is critical for users of NA-based tools to recognize that primers and probes do not
exhibit perfect specificity for the target pathogen—and exclusively that pathogen—in
every assay
• . In the clinical micro biology lab, collection of an appropriate sample is regarded as
“the most important step in the testing process and the same is true for plant disease
diagnosis.
• Contamination risks. Because the amplification of target DNA that occurs in PCR is
exponential, the technique is extremely sensitive to contamination .
• PCR inhibition. Organic and inorganic compounds present in host tissues, compo
nents of microbial cells, and other materi als can all inhibit PCR

24. CONCLUSION
Nucleic acid based detection methods for plant viruses offer
sensitive,specific and rapid detection,crucial for early disease
diagnosis and management.they provide valuable tools for plant
pathologist,breeders,and farmers to monitor and control viral
infection,ultimately contributing to sustainable agriculture and food
security.

25. REFERENCE;-

26. THANK YOU!
any querries??