disease diagnosis, types of diagnostic kits, nucleic acid based that include pcr, rt pcr, microarray, protein based which include ELISA, types of elisa, comparision among all types of diagnostic kits

1. Development of
diagnostic kits
Presented by:
Shiney Chatak

2. Introduction
• Annual losses by diseases: ˃42%
• Diagnosis of plant disease is to identify the disease nature and to
determine the causal agent whether living or non living
• Traditional method -: Visual examination. possible only after major
damage has already done
• To save plants from irreparable damage by pathogens, infection should
be identified even before it becomes visible that can be reached by
using very sensitive methods for early diagnosis of disease

3. Is early detection possible?
• An attack by pathogen generates a complex immune response in plant
resulting in production of disease-specific proteins involved in plant
defence and in limiting the spread of infection
• Pathogens also produce proteins and toxins to facilitate their infection,
before disease symptoms appear.
• These molecules play vital role in the development of plant diagnostic
kits.

4. Diagnostic kits
• Means rapid method for the detection of microorganisms
• Designed to detect plant diseases early, either
by identifying the presence of the pathogen in the plant (by testing for
the presence of pathogen DNA), or
by molecules (proteins) produced by either the pathogen or the plant
during infection

5. Advantages
• Minimal processing time
• More accurate
• Some require laboratory equipment and training, other procedures can be
performed on site by a person with no special training

6. Crops for which diagnostic kits are used
• Rice
• Potatoes
• Papaya
• Tomatoes
• Banana
 Also used for detecting genetically modified organisms (GMOs)

7. Types
• Nucleic acid Based diagnostic kits
• Protein Based diagnostic kits

8. Nucleic acid-Based Diagnostic Kits
• Based on the ability of single stranded nucleic acids to bind to other
single stranded nucleic acids that are complementary in sequence
(referred to as homologous)
• Tool used in these diagnostic kits: Polymerase Chain Reaction (PCR)

9. PCR (Polymerase Chain Reaction)
• 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

10. Principle of PCR
• 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

12. Real time PCR
• Real-time PCR (RT PCR) follows the general principle of polymerase chain
reaction
• Key feature -: amplified DNA is quantified, using fluorescent dyes, as it
accumulates in the reaction mixture after each cycle.
• Offers several advantages over normal PCR, including:
Reduced risk of sample contamination
Provision of data in real time and simultaneous testing for multiple pathogens.
Rapid on site detection
Require fewer reagents
• Real-time PCR protocols are among the most rapid species-specific detection
techniques currently available.

13. • 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

14. Schematic diagram of RT-PCR procedure

15. GRAPH OF RT-PCR

16. DNA Microarray
• 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 labelled complementary
sequence provides a signal that can be detected and analysed
• Nanochip are type of microarray
• 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

17. • DNA microarrays are also of great use for simultaneous pathogen
detection
• Important, as plants are often infected with several pathogens forming
disease complex
• Consist of pathogen-specific DNA sequences immobilized onto a solid
surface
• Sample DNA is amplified by PCR, labeled with fluorescent dyes, and
then hybridized to the array

18. Microarray analysis

19. Diseases for which PCR KITS have been
developed
• Black Sigatoka disease (Mycosphaerella musicola) in bananas
• Late blight (Phytophthora infestans) in potatoes
• Fusarium wilt (Fusarium oxysporum f. sp. Vasinfectum) in cotton

20. Advantages over other
• Very sensitive compared to other techniques; detection of a small
amount of DNA is possible
• Help to detect the presence of pathogens that have long latent periods
between infection and symptom development
• Can quantify pathogen biomass in host tissue and environmental
samples, and at the same time detect fungicide resistance
• Primers in PCR diagnostic kits are very specific for the genes of a
pathogen, and amplification will occur only in diseased plants

21. PCR-based Diagnostic kits reaction

22. Disadvantages over other
• Expensive compared to protein-based diagnostic methods
• Requires costly equipment's
• False positive results due to contamination from operator,
residual matter in testing utensils or air contamination can
result in false positive reaction

23. Protein-Based Diagnostic Kits
• First step in a defence response reaction is recognition of an invader
by a host’s immune system
• This recognition is due to the ability of specific host proteins, called
antibodies, to recognize and bind proteins that are unique to a
pathogen (antigens) and to trigger an immune reaction

24. • Protein-based diagnostic kits contain an
antibody (the primary antibody) that
can either recognize a protein from
either the pathogen or the diseased
plant.
• Because the antibody-antigen complex
cannot be seen by the naked eye,
diagnostic kits also contain a secondary
antibody, which is joined to an enzyme.
• Enzyme will catalyze a chemical
reaction that will result in a colour
change only when the primary antibody
is bound to the antigen.
• Therefore, if a color change occurs in
the kit’s reaction mixture, then the plant
pathogen is present,

25. • Enzyme-linked
immunosorbent assay
(ELISA) method makes use of
this detection system, and
forms the basis of some
protein-based diagnostic kits.
• ELISA kits are very easy to
use because test takes only a
few minutes to perform, and
does not require sophisticated
laboratory equipment or
training

26. ELISA
• Enzyme-linked immunosorbent assay
• First described by Engvall and Perlmann in 1971
• Uses a solid-phase type of enzyme immunoassay (EIA) to detect the
presence of a protein in a liquid sample using antibodies directed against the
protein to be measured
• Process:
Antigens from the sample to be tested are attached to a surface.
Then, a matching antibody is applied over the surface so it can bind the
antigen.
This antibody is linked to an enzyme and then any unbound antibodies are
removed.
In the final step, a substance containing the enzyme's substrate is added
If there was binding the subsequent reaction produces a detectable signal,
most commonly a colour change

27. ELISA kit

28. • First ELISA kit developed to diagnose plant disease was by the
International Potato Center (CIP) to detect the presence of all races,
biovars, and serotypes of Ralstonia solanacearum (bacterial wilt or
brown rot in potato). Their other kits:
• sweet potato viruses: SPFMV (sweet potato feathery mottle virus),
SPCSV (sweet potato chlorotic stunt crinivirus), SPMSV (Sweet
potato mild speckling virus), SPMMV (Sweet potato mild mottle
virus), SwPLV (Sweet potato latent virus), SPCFV (Sweet
potatochlorotic fleck virus), SPCaLV (Sweet potato caulimovirus), and
C-6 (new flexuous rod virus)

29. Types of ELISA

30. ELISA based Kits available in market for
plant diseases
• Ratoon stunting disease of sugarcane
• Tomato mosaic virus
• Papaya ringspot virus
• Banana bract mosaic virus
• Banana bunchy top virus
• Watermelon mosaic virus
• Rice tungro virus

31. Comparison of different Diagnostic kits

32. Future perspectives
• Diagnostic kits are an investment: they may be expensive, but the costs can
be offset by gains, such as reduced crop losses and more environment-
friendly crop-management practices
• Their development should be made a priority by both the public and private
sectors in developing countries
• Genetic Engineering Services Unit of Egypt’s Agricultural Genetic
Engineering Research Institute has developed diagnostic kits and testing
services to detect viruses in crop plants.