VPB 321: Animal Biotechnology (2+1)

APPLICATIONS OF PCR IN DISEASE DIAGNOSIS

• PCR has many exciting varied applications:
• PCR can be used to amplify a specific gene present in different individuals of a species and even in different somatic cells or gametes say humans sperms. These copies can be used for cloning.
• Alternatively they can be sequenced to obtain information on the mutational changes in gametes in genes of different individuals, cells or gametes such data can be used in disease diagnosis, population genetics, estimation of recombination frequencies.
• PCR permits early diagnosis of malignant diseases such as leukaemia and lymphomas. Which is currently the highest development in cancer research.
• PCR assays can be performed directly on genomic DNA samples to detect translocation specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.
• PCR also permits identification of non-cultivable or slow growing micro organisms such as Mycobacteria , Anaerobic bacteria or viruses from tissue cultureassays and animal models.
• The basis of PCR diagnostic application is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.
• Viral DNA can like wise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus and the PCR can be used for diagnostic analysis or DNA sequencing of the viral genome. The high sensitivity of PCR permits viral detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in the treatment . The amount of virus (viral load) in a patient can be quantified by PCR based DNA quantification techniques.
• Tumor calls shed from solid primary tumors can be detected by the PCR based on the selective amplication of mutated tumor genes expressed in tissue specific manner when tumor specific alterations are amplified, few tumors cells can be detected in excess of normal cells derived from the same tissue. Thus malignant cells can be detected specifically in pancreatic juice, urine, sputum.
• Viruses:
• Human retroviruses eg: Human Immuno deficiency virus types 1 and 2 and Human T- cell Lymphotrophic virus types 1 and 2 replicate through an RNA intermediate.
• The RNA intermediate can be detected after performing a reverse trancriptase step to generate a DNA template for PCR.
• As a consequence of replicative cycle and the reduced fidelity of the viral-replicative polymerase and the reverse transcriptase enzymes,considerable heterogenicity between viral isolates is seen. So it is necessary to target highly conserved regions of the viral genome for a PCR assay.
• Othert clinically important viruses:
• Hepatitis B virus (HBV)
• Human papilloma virus (HPV).
• Bacteria: Diseases caused by salmonella, shigella and vibrio mainly by faecal contamination of water and the reserve of the bacterial pathogens in the conditioning tanks such as legionella pneumophilia, causing legionnaires disease are of concern to be monitered by the way of PCR amplification of a target gene sequence followed by hybridization with specific gene probes provides both specificity and sensitivity for the monitering.
• Yet another is the use in the detection of bacterial antibiotic resistance such as bacterial antibiotic resistance such as methicillin resistance in the staphylococcus aureus using primers specific for these genes.