DNA probes are pieces of radiolabelled or chromogenically labeled pieces of single-stranded DNA that will bind to DNA that is complementary to the probe using hybridizing technique. The specificity of the interaction in base pairing during DNA or RNA synthesis enables the production of specific DNA probes.
DNA or genetic probes can be used like antibodies as sensitive and specific tools to detect, locate and quantitate specific nucleic acid sequences in clinical specimens. Individual species or strains of an infectious agent can be detected even if they are not growing or replicating because of the specificity and sensitivity of DNA probe techniques.
Development of nucleic acid probe
All microorganisms, simple or complex, contain some unique sequences of DNA or RNA within their genome that ‘distinguish them from all other organisms. DNA probes are chemically synthesized or obtained by cloning specific genomic fragments or an entire viral genome into bacterial vectors (plasmids, cosmids).
Hybridization is the process whereby two single-strands of nucleic acid come together to form a stable double-stranded molecule.
Detection of hybridization:
Hybridization assays require that one nucleic acid strand (probe) originates from an organism of known identity and the other strand (the target) originates from unknown organisms to be detected or identified. The sample serves as a source of nucleic acid to be analyzed and can consist of a suspension of an unknown organism (for culture confirmation) or a clinical specimen such as sputum or stool
Identification of unknown organism is established by positive hybridization (i.e. duplex formation)
between a probe nucleic acid strand (from unknown organisms) and a target nucleic acid strand from the organism to be identified. Failure to hybridize indicates lack of homology between probe and target nucleic acid. Positive hybridization identifies the unknown organism as being the same as the probe-source organisms. With a negative hybridization test, the organism remains undetected or unidentified
Applications of Nucleic Acid Probes
DNA probes have already been used successfully to identify a wide variety of pathogens, from simple viruses to pathogenic bacteria and parasites.
1. Antibiotic resistance: Probes have also been developed which can recognize specific antibiotic resistance genes, so that, antimicrobial susceptibility of an infecting organism can be determined directly
without primary isolation and growth.
2. Culture confirmation: DNA probes are being used for culture confirmation as an alternative to
conventional, time-consuming or labor-intensive methods in the diagnostic laboratory. For example,
DNA hybridization makes it possible to rapidly identify Mycobacterium tuberculosis, M kansasii, M
avium complex, and M gordonae isolated in culture, significantly reducing the time for reporting of the
species of the isolate
3. Direct detection in clinical specimen: For detection of fastidious organisms directly in clinical
specimens probe technology may also be used.
Examples are Neisseria gonorrhoeae and Chlamydia trachomatis.