Biotechnology and Bioinformatics (1+1)

4.2.9. DNA – based diagnostics

Molecular biology has been used to design a new gene ration of diagnostic tools, the PCR ( Polymerase Chain Reaction) and Gene Probe s. The key to DNA-based diagnostics is the generation of the target pathogen through recombinant DNA technology . This is done by purifying the infectious agent of interest and isolating its nucleic acid. The isolated DNA fragment has to be sequence d. Once the adequate genetic information (sequence information) is generated, the information can be used in PCR or gene probes.

Presently, PCR methods are available for the detection of many pathogens of shrimp:

Vibrio vulnificus (Hill et al., 1991);

V. parahaemolyticus (Karunasagar et al., 1997);

V. penaeida (Genmoto et al., 1996);

MBV (Lee et al. 1993);

IHHNV (Lightner et al., 1994);

rod shaped nuclear virus of P. japonicus (Takahashi et al. 1996), and BP (Wang et al. 1996).

PCR has been used to detect pathogenic bacteria and viruses in hatchery and aquaculture situations (Winton, 1992).

Nucleic acid probes are segments of DNA or RNA that have been labeled with enzymes , antigenic substances, chemiluminiscent substances or radioisotopes. Probes can be directed against either DNA or RNA targets.

Probes bind with complimentary sequences of pathogenic DNA during the detection process providing a signal (like colour change) that can be identified or measured.

• Today, non-radioactive probes (e.g. digoxigenin (DIG) labeled probes) are gaining importance due to their high level of sensitivity and safety compared to radioactive probes.
• In-situ hybridization and dot blot hybridization are gene probes being used in aquatic disease diagnostics. However, PCR has definite advantages over gene probes in its sensitivity for direct detection in clinical specimens.

Nucleic acid hybridization reaction consists of four components;

i) the probe,

ii) the target DNA/RNA (in the sample),

iii) the reporter molecule (the label on the probe), and

iv) the hybridization method.

• Hybridization can be performed in solutions or on solid support (dot-blot) or even on sections of tissue fixed on slides (in-situ hybridization).
• In-situ hybridization has the advantage in that non-specific tissue effects which may result in false positive diagnosis in dot-blot assay can be distinguished from specific histological lesions (Lightner, 1996).
• Presently, in shrimp disease diagnosis, hybridization probes are available for many viruses such as Infectious Hypodermal and Haematopoeitic Necrosis virus (IHHNV), Hepatopancreas Parvo-like Virus (HPV), Baculovirus Penaei (BP) and Monodon baculo virus (MBV).