Nucleic acid disease diagnosis of fish

1. Nucleic Acid Based Disease
Diagnosis
Jayanta SubhashTiple
B.F.Sc. 3rd year.
Enll.No.F/15/061
College of Fishery Science,Udgir.dt.Latur

2. • Nucleic acid based diagnosis is highly specific, sensitive and
rapid method widely used in health management.
• The detection is mainly based on structure and sequence of
bases of nucleic acid.
• Nucleic acid based diagnosis is becoming common and popular
in fish and shellfish health management

3. Commonly employed nucleic acid based
diagnosis in fish two way:
• A. DNA hybridization
• B. Polymerase chain reaction (PCR)

4.  Nucleic acid are made up of nitrogen base .
 The primary sequence of bases in DNA or RNA have region
which are unique for a particular organisms or gene.
 The sequence of DNA is composed of series of four
phosphorylated base, i.e. Adenine, thymidine, guanidine, and
cytosine.

5. Principle :
Nucleic acid probe are segments of DNA or RNA that have been
labeled with enzymes, antigenic substances and
chemiluminescent moieties or radioisotopes.
They would bind with high specificity to complementary
sequences of nucleic acid to form double stranded molecules and
this process is called hybridization.
DNA hybridization:

6.  Targets and probes
• DNA hybridization whether done on solid phase, in suspension or in situ.
• The Target: - Target is the sequence of DNA that is to be detected.
• The Probe: -A DNA sequence labeled with a radioactive element used to
identify the position of a segment with the complementary sequence by
binding to it.
 Characteristics of probe
• highly radioisotopes labeling of probe with S35 , P32 , H3 and H or other
radioactive elements to the target strand.
• The probe length of 20-100 base pair is the most common.

8. Two method of DNA hybridization:
1 .DNA hybridization on solid phase .
2. In situ DNA hybridization .
• 1 .DNA hybridization on solid phase:
 In this method the suspected fish/shellfish sample is
homogenized and dotted on to a nylon membrane or nitrocellular
paper and DNA is exposed from protein usually by digesting with
trichloroacetic acid.

9. DNA in the sample is then denatured at appropriated temperature and
hybridized at appropriated temperature with a probe linked to an
isotope or enzyme.
Probe hybridization is detected by autoradiography or with a chromogen
substrate.
This protocol has been adopted to detect DNA in large number of sample
to developed a dot blot hybridization.
The method is ideal for diagnosis and also for genotyping of pathogen.

11. • 2. In situ DNA hybridization:
• In this technique histological section of the suspected sample are
employed.
• DNA of the pathogen in the tissue is exposed using tricholroacetic
acid and hybridized with isotope or enzyme labeled probe.
• Hybridization is detected by autoradiography or enzyme substrate
reaction .
• The technique is ideal for diagnosis and studying site of replication in
the target organs of the host.

13. Polymerase chain reaction (PCR)
• PCR is an in vitro technics,by which a specific sequence of a DNA
molecule can amplified by repeated enzymatic thermocyclic prosses .
• PCR technique was invented by kary.b mullis in 1985.
• Major steps in PCR
• 1. Denaturation
• 2. Primer annealing
• 3. Extension

16. General PCR protocol used for detection of viral
pathogen in aquaculture
• The protocol has several steps such as collection of sample, extraction
of DNA amplification of DNA and detection of the amplified DNA.
1. Sampling : fresh samples should be collected and fixed immediately
in alcohol. Appropriate sample size and sampling strategy are
necessary to avoid false negative result.
2. DNA extraction : samples are subjected to DNA extraction by
suitable method ( ex. Alkaline lysis method)

17. 3.DNA amplification : target DNA is amplified with a cocktail of taq polymerase
oligonucleotide primer and nucleotides in a thermal cycle in 20-30 cycle. Each
cycle consist of denaturation at 1000c for 1 min and extension at 700c for 2min
• If required 2 step PCR carried out by using 1-5%of amplified DNA from the
above 1 step . in 2 step PCR is amplification of DNA can be performed either
with the same set of primer or internal primer
4.Detection of amplified DNA :the amplified DNA along with marker is
subjected to electrophoresis in a 1.5% polyacrylamide gel the gel is then stained
with ethidium dibromide and fluorescing DNA bands under u.v light are
recorded. Positive and negative controls should yield appropriated result in each
test for validation of the DNA amplification.
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18. THANKS FOR KIND ATTENTION....
FINFISH AND SHELLFISH DISEASES AND MANAGEMENT