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Identification of Foodborne Bacterial Pathogens by Gene Probes

Saturday, July 10, 2010

DNA Hybridization

The identification of bacteria by DNA probe hybridization methods is based on the presence or absence of particular genes. The physical basis for gene probe tests arises from the structure of DNA molecules themselves.

DNA composes of two strands of nucleotide polymers that form a double helix held together by hydrogen bonds. The hydrogen bonds holding the strands together can usually be broken by raising the pH above 12 or the temperature above 95°C. As a result, a single-stranded molecule is achieved and the DNA is considered denatured. At a lowered pH or temperature, the hydrogen bonds are reestablished, reforming double-stranded DNA. The source of the DNA strands is inconsequential as long as the strands are complementary. If the strands of the double helix are from different sources, the molecules are called hybrids and the process is termed hybridization.

A gene probe composes of double-stranded DNA. It has either an entire gene or a fragment of a gene with a known function. The short pieces of single-stranded DNA can be synthesized based on the nucleotide sequence of the known gene, commonly known as oligonucleotides. Both natural and synthetic oligonucleotides are used to detect complementary DNA or RNA targets in samples.

Double-stranded DNA probes are required to be denatured before the hybridization reaction beginsThe target nucleic acids are denatured by high temperature or high pH, after which labeled gene probe is added. If the target nucleic acid in the sample contains the same nucleotide sequence as that of the gene probe, the probe will form hydrogen bonds with the target. The bound label signals the presence of probe-target complexes.

The oligonucleotide of known sequence, derived from gene of known function, is end-labeled with radioactivity. DNA probe is allowed to incubate with DNA extracted from a sample. If target DNA contains sequences complementary to those of the probe, the probe and its radioactive label will bind with the sample DNA.

Colony Hybridization

DNA hybridization tests may be performed in many ways. One way is by the colony hybridization assay.

Firstly, a portion of a homogenized food is spread-plated on an appropriate agar. After incubation, the colonial pattern is transferred to a solid support (a membrane or paper filter) by pressing the support onto the agar surface. The cells are then lysed by high pH and temperature, which denatures and attaches the DNA to the support. The solid support with the attached target DNA is incubated with a labeled probe.

The radioactive probe DNA that is bound to the target on the support is often detected by autoradiography. Radioactive decays expose the X-ray film, which is placed over the support. When developed, black spots appear where cells are harboring the same gene as the probe.

If an enzyme-labeled probe is used, a chromogenic substrate is added. A colored spot will develop if the probe-associated enzyme is present. Each spot represents a bacterial colony that has arisen from a single cell.

The number of cells harboring the target gene in the original sample can be calculated by multiplying the number of spots by the dilution factor.

Source:

U.S. Food and Drug Administration. (2001, January). Identification of Foodborne Bacterial Pathogens By Gene Probes. website:
http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm072659.htm