Conventional diagnostic techniques

Topic - Disease diagnostic kits

Welcome, to the last talk of the week that is disease diagnostic kits. We have seen that in the
previous talk that we have conventional methods as well as certain molecular methods for
detection and diagnosis of certain plant pathogens whether it is seed borne in nature or that is
associated with any sort of plant diseases, but with the advancement in plant biotechnological
tools and techniques people are now able to have their Disease Diagnostic kits which are very
handy and ready to use and they are able to detect the possible causal organisms of the plant
diseases very efficiently in a short span of time.
So biotechnology has allowed the development of Diagnostics which is assisted farmers
worldwide in managing different diseases affecting their crops. New diagnostic techniques
require minimal processing time that is very important and they are more accurate in
identifying the pathogens. So this is how this diagnostic kits are becoming popular day by day.
This Diagnostics are based on rapid detection of proteins or DNA that are specific to each
pathogen, disease or condition. So they are designed to detect different pathogens or different
conditions separately. Some procedures require laboratory equipment and training while other
procedures can be performed on site by a person with no special training. So it is the device
that gives us the indication or they tells us whether that particular pathogen is present or not
on the sample. So there is no need of highly trained personnel to be involved in such diagnostic
kits, associated with plant disease diagnosis.
So plant disease diagnostic basically based on the principles of protein or DNA based methods
and there is a broad range of ELISA that is protein based diagnosis based method that is
available. ELISA kits are available for plant pathogen detections and with very high test
performance characteristics to allow accurate rapid simple and high-throughput identification
of the organisms that causes plant disease. The immunological technique based on ELISA kits
offer considerable advantage over traditional diagnostic methods and PCR-based techniques as
these Eliza kids are easy to handle and very fast to give the results, within few minutes one can
have the results in hand. In addition a wide range of ELISA-based rapid test strips are also
available with visible color chain signal by using lateral flow devices which are diagnosed for on-
site robust and fast detection of plant pathogens by even unskilled personnel.
That is the beauty of this ELISA kits and they are able to detect plant pathogens very efficiently.
Some of the examples of diagnostic techniques like (enzyme linked immunosorbent assay) kits.
So, ELISA kits are based on the ability of an antibody to recognize a certain protein substance or
antigen associated with a plant pathogen. So pathogen specific antibodies are developed and
these antibodies are used for rapid detection of pathogens in this ELISA kits. The kits are very
easy to use and can be used in field to disease only in detected disease only in 5 minutes. In
addition they do not require sophisticated laboratory, equipment or training. There are already
numerous ELISA test kits available in the market to detect diseases of root crops, ornamental
crops, fruits and grains and vegetables. So technically the method involves collection of infected
plant 04:01 part and this is then macerated in the tube with the solution that is buffering
solution and the resultant solutions a suspension is then pipetted it out and it is then a few
drops are placed in the well of the diagnostic strip and appearance of a single line gives the
positive indication of the pathogen because this line is basically, it is the antibody and once the
antigen that is protein, specific protein is of the pathogen moves through this lateral flow
device and reaches the antibody then there is development of color and in case, if pathogen is
not present then there is no development of such strip success line is observed. And, this is how
detection of pathogens specific in nature can be obtained by using these strips and even the
people with little knowledge can establish whether the plant sample is having that particular
pathogen associated or not. So for different pathogens we have different diagnostic or, little
flow devices such as for Erwinia amylovora Phytophthora then Phytophthora species Raistonia
solanacearum and so on. So there are different kits available for different pathogens but more
or less the basic technology is same for detecting all the pathogen except for the antibody used
for different strips are different for detection of different pathogens. Then there is another
technology known as ImmunoComb, this is from agdia company and it joins together the most
requested Agdia test for plant virus detection because the grower can test for Cucumber
mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and
Tobacco mosaic virus(TMV) virus all at one time. The procedure ImmunoComb involves the
samples are grounded in a special bag that allows for easy extraction the comb of four test
strips is then easily placed into the sample and the test takes just minutes to form clear yes or
no results. So in the sample four different strips are inserted and if any of this four pathogen is
present in the sample then one of the strips will give color indication for presence of the one
specific viruses and that's how within minutes one can confirm whether the particular virus is
present or not.
Then there are direct tissue blotting method - in this technique utilizes specific antibodies to
detect the presence of plant pathogens. These tissue samples are pressed to draw out proteins
onto a special paper and the antibodies are added to the sample. The color inducing reagent is
added afterwards had to react with the antibody pathogen complex. And color reaction
indicates a positive result and pinpoints the location of the pathogen in the diseased tissue. So
what is done in the direct tissue blotting method is that one has to squeeze out the juice of the
infected tissues and it should be spotted on a specific paper and then antibody is added along
with the colouring agent. If the color is produced then it can quickly detect whether that
particular pathogen or again switched antibody is raised is present or absent.
Then DNA and RNA probes - this is another set of tools that can be used in plant disease
diagnostics using nucleic acid whether it is DNA or RNA as probes. These probes are fragments
of nucleic acid arranged in sequence complementary to that of DNA or RNA of the pathogen.
Because of the sequence complementation each other the probes can be used to identify
specific Disease. So pathogens DNA or RNA specific probes can be applied to detect or action of
the pathogen DNA or RNA in the sample and because of their complementary they will emit
different types of signals and these signals will then confirm whether that particular pathogen is
present or absent. Then Squash blot method - this is another method where the tissue from an
infected plant is “ squashed” into a specialized piece of paper we call it a membrane. Then the
membrane is treated with a probe that can bind with the DNA or RNA of the plant pathogen
suspected to be in the tissue. Binding will occur when complementary sequences are present.
After adding several more substances to the membrane a color reaction indicates that the
probe and the pathogen DNA/RNA have bound to each other and the disease is present. No
color reaction means the test is negative for the particular disease.
So this is another method very rapid and quick method where the tissue is squashed into a
membrane and then DNA or RNA probe is added to the membrane. The pathogen
complimentary pathogen DNA or RNA is present in the squashed area then the DNA probe will
go and bind with it. Then Polymerase Chain Reaction in short we call it PCR. It is used nucleic
acid probes to detect the presence of a pathogen. But this is lot more sensitive compared to
other techniques as PCR can detect very small amount of pathogens genetic material or sample
and amplify certain sequences to a detectable level. PCR can be used to detect the presence of
pathogens in air, soil and water. Spores, especially those produced by fungi are the primary
source of infection to initiate epidemics. So farmers can therefore keep track of the pathogen
and apply the necessary control to prevent the spread of the disease. So it's another highly very
sensitive instrument polymerase chain reaction and this technique is used to detect even very
small amount of pathogen sample that is present either in air, soil or water so, that the farmers
can be aware of the trick that is coming in short time. So Pocket Diagnostics have developed
rapid test strips for plan disease in the diagnosis. And this lateral flow rapid test strips normally
are available for detection of different plant pathogens.
We need just need to cut or tear sample into small pieces and put into bottle containing buffer
and small bearings. Here instead of using a rod the tissue is macerated by using ball bearings by
shaking it firmly for 30 to 60 seconds to break the sample tissue and allow the liquid to settle
down. Then draw the liquid with the help of a pipette. It should be avoided the debris and air
bubbles. Keeping the test device level add two drops of the sample well into the device. Here
they have added the pipetted out the sample two drops and the results can be obtained within
10 minutes as we have discussed earlier were in a positive interaction give rise to a new signal
that is a new band on the diagnostic kit.
Then PCRD is another technology where one can have rapid detection of plant pathogen
following PCRD methodology. After the PCR polymerase chain reaction for detection of
amplification we have to go for agarose gel electrophoresis but this particular diagnostic kit can
help us to avoid that gel electrophoresis and thereby reduce the time to a significant amount
for detection of the plant pathogen that is associated with the disease sample. So in PCRD is
just a simple and rapid alternative to gel electrophoresis that can be performed in a matter of
minutes without the need for expensive equipment or exposure to intercalating dye and UV
light. These are also very harmful to human being and in this with the use of PCR D we can help
us how to avoid this dyes and UV light PCRD is a basically a nucleic acid lateral flow amino acid
suitable for use with PCR, LAMP technology then, RPA technology as well as HDA technology.
The format of PCRD is suitable for use in both high-throughput laboratories as well as small
field based laboratories.
Then what are the benefits of Diagnostic Kits we have already seen the lot of benefits of the
diagnostic kits but there are two important benefits that can be derived from the use of
diagnostic kits and that is in field decision making process. They help us in In-field decision
making process and they reduce the cost per sample of analysis. So they helped us to rapid test
in the field within minutes, this then enables the commencement of the management
strategies quicker than if a sample was sent off to lab which is an obvious benefit for yield. So it
saves time and quick detections means easy and immediate recommendations for
management of the problem that is associated in the field, because sending the sample to lab
means time is more consumed in the process and to get back the desired result and they are
normally cheaper in nature because of no need to send the samples to the lab. Whereas, using
a rapid test means the cost per sample can be reduced because, they are specifically designed
for this specific pathogens and they can be easily handled by all kinds of personalities that are
associated for plant disease diagnosis. So with this we have come to an end of the second week
of this course Detection, Diagnosis and Management of plant diseases and in the next week we
will be talking about the conventional methods of plant disease diagnosis and for aware of the
tools and techniques that has been basically used for plant disease diagnosis. We will be looking
into those tools and techniques in a more elaborated way in the following weeks.

Thank you very much