VPB 321: Animal Biotechnology (2+1)

APPLICATIONS OF DNA PROBE/DNA HYBRIDIZATION IN DISEASE DIAGNOSIS

• The application of Insitu PCR has been mainly confined to two areas foreign gene detection and identification if gene alterations, although many other areas remain to be fully explored because of its extremely high sensitivity. Insitu PCR for DNA is limited to the detection of gene sequence that are not normally present in the tissue samples of interest.
• Insitu PCR is a new molecular technique that combines the extreme sensitivity of PCR with the cellular localization provided by In situ hybridization(ISH). It is based on the principles of amplification of gene sequences within intact cells or tissue sections in order to increase copy number to levels detectable by ISH.
• Insitu PCR can be used in detecting viral infection. In case of HIV one can detect the extent of infection and the effects of treatment. One can utilise in gene therapy for verification of integration and expression of a desired gene in vivo.
• Insitu can also be used to determine tumour burden before and after chemotherapy where specific genetic abberants are associated with certain types of malignancy or to determine pre neoplastic by examining P⁵³ mutations.
• They can be used to identify infectious agents, tumours, marker genes, cytokines growth factors and their receptors as well as in gene therapy. It can be used to do immuno - histo chemistry together with RNA and DNA amplification at a single cell level.
• The successfully amplified and detected are
  • Human immuno deficiency virus(HIV-1)
  • Simian immunodeficiency virus(SIV)
  • Human papilloma virus(HPV)
  • Hepatitis B virus(HBV)
  • Cytomegalovirus(CMV)
  • Epstein- Barr virus(EBV)
  • Human-herpes virus-6(HHV-6)
  • Herpes simplex virus(HSV)
  • Lymphogranuloma venereum(LGV)
• P⁵³ and its mutations, mRNA for surfactant protein A, estrogen receptors, inducible nitric oxide synthatase(INOS) gene sequences associated with multiple sclerosis in various tissues including peripheral mono nuclear cells, lymph nodes, spleen, brain, skin, breast, lungs, cytological specimens, tumours, cultured cells, tissues.
• It is a good tool to assist gene therapy in tracing the whereabouts of foreign genes and pinpointing the exact locations of introduced gene sequences after gene therapy delivery.

Applications of PCR and DNA probes for animal disease diagnosis

S.No.	Species	Disease	Target Gene
1.	Cattle	Mycobacterium bovis Haemorrhagic septicaemia Clostridium chauvoei Foot and Mouth disease	IS1081 16S and 23S rRNA 16S rRNA C-coding sequence
2.	Sheep & Goats	Mycobacterium avium subspecies paratuberculosis Staphylococcus aureus Brucella Sheep pox virus Pestivirus Babesia ovis Theileria Toxoplasma gondii	IS900 se a, b, se c & d gene sequences IS711 Inverted Terminal Repeats 5’-UTR Small subunit ribosomal RNA Small subunit ribosomal RNA B1 gene
3.	Dog	Canine distemper Canina parvo Rabies	NP gene VP1/VP2 Nucleoprotein gene
4.	Poultry	Newcastle disease Infectious bronchitis Infectious bursal Marek’s disease	Fusion gene Nucleo protein gene VP gene Meq gene