Detection of Parasite DNA by using PCR and suitable primers. Polymerase Chain Reaction

1. DETECTION OF BABESIA
CANIS BY PCR ASSAY
Sourabh Gurav
Neha Patil
Anjali Pawar

2. Babesia Canis
• Infects Red Blood cells
• Transmitted by brown Dog ticks
• Ticks transmit sporozoites of B. canis to the dog which later enters
RBCs
• Sporozoites loses outer covering inside the cell & turns into
merozoites
• This follows with the sexual reproduction and latter asexual
reproduction forming multiple sporozoites

3. Laboratory Diagnosis
1. Microscopic analysis:
• Microscopic Examination of Thick and thin blood smears
• 100 X oil immersion magnification under microscope
• Wright – Giesma stain
2.Fluorescent Dyes
• Differential staining of nuclear DNA in green and cytoplasmic RNA in red
• Kawamoto Technique
• Fluorescent microscope or light microscope adapted with an interference filter system.
3. Molecular Diagnosis
Amply a fragment of the 18s rRNA gene using PCR primers specific at genus level and
perform DNA sequencing of amplicon to determine species.

4. • Inclusion bodies in RBCs
seen under microscope
ELISA (Enzyme Linked Immunosorbent
Assay) : using parasite as an antigen and
specific antibodies against it that are
fluorescently labelled

5. Isolation Of DNA

6. PCR (Polymerase Chain Reaction)
• Invitro DNA Replication process
• Amplifies small amount any genetic material
• Repetitive cycles of denaturation, annealing and elongation
• PCR mixture : dNTPs, Buffer, Taq Polymerase, Template DNA
• Thermocycler Machine
• Theoretical Number of DNA molecules obtained= 2n
n= number of cycles

7. • Stages of target DNA replication(One cycle)
• Temperature Profile of the process in a thermocycler for amplification
of target DNA

8. Primers
• Primers : short single stranded Nucleic Acid or oligonucleotides
• Customized to anneal to specific site
• Characteristics of primers
1. Length (18-22 nucleotides)
2. Primer melting Temperature (52-580 C)
3. Primer Annealing temperature
4. GC content (40-60%)
5. GC clamp

9. Agarose Gel electrophoresis
• Apparatus & Chemical: Comb, Agarose gel, Buffers, Battery, electrode
• Gel matrix (works as Sieve or netting)
• DNA loaded in gel migrates through a voltage gradient
• DNA : negatively charged molecule
• Smaller molecules migrate quickly while Larger molecule migrate
slowly
• Gel is placed in a container with the conductive, pH controlled buffer
solution (Tris -acetate- EDTA)

10. Visualisation of DNA in Agarose gel
• Stain used to visualize DNA : methylene Blue or Ethidium Bromide
• Gel documentation System / Gel doc : to capture images of agarose
gels
• UV or Blue light illuminators excite fluorescent stains that is captured
by camera

11. Separated DNA fragments (Lane A B C and D) and DNA ladder (Extreme Right) on agarose
Gel Electrophoresis