Detection of Babesia canis by PCR assay
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This document describes the detection of Babesia canis by PCR assay. It discusses how B. canis infects red blood cells and is transmitted by brown dog ticks. Laboratory diagnosis can be done through microscopic analysis of blood smears, fluorescent dyes, or molecular diagnosis using PCR to amplify the 18s rRNA gene and perform DNA sequencing to identify the species. The document outlines the PCR process, including isolation of DNA, use of primers, agarose gel electrophoresis to visualize amplified DNA fragments.
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This document discusses molecular diagnostic techniques used in pathology. It describes common techniques like PCR, blotting methods including Southern blot, Northern blot and Western blot, and hybridization techniques such as in situ hybridization and FISH. These techniques allow manipulation and analysis of DNA, RNA and proteins and have applications in neoplastic disorders, infectious diseases, inherited conditions and identity determination. The document provides details on the principles, requirements and procedures of various molecular diagnostic techniques and their uses in hematological and non-hematological malignancies, infectious diseases, inherited genetic disorders and identity determination.
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- 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
- 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