Molecular Probes Can Be Specific and Highly Sensitive

Although tests that require pathogen growth are often easy to perform, they can require considerable time; consequently, physicians frequently prescribe treatment without growing the pathogen and learning the cause of disease. This lack of precision is being corrected by replacement of conventional agar-plate methods with rapid, sensitive nucleic acid tests. For these tests, nucleic acids are extracted from diseased tissue or blood samples, and then they are examined for the presence of a particular pathogen nucleic acid. With DNA, detection begins by forcing apart the two strands of DNA from a patient sample. (Boiling a DNA solution is sufficient to separate the strands, and rapid cooling keeps them from coming back together.) The sample is mixed with a single-stranded DNA probe that is pathogen-specific. Incubation under proper conditions enables the nucleic acid from the laboratory sample, the probe, to bind with single-stranded pathogen DNA obtained from the patient sample. The result is a double-stranded hybrid DNA if the patient sample contains DNA with nucleotide sequences complementary to those in the probe. Formation of duplex DNA containing single-stranded nucleic acids from different sources is called nucleic acid hybridization. Because hybridization occurs only when the nucleotide sequences are complementary, hybridization serves as a specific test for a particular pathogen species. Probes called molecular beacons are available that emit fluorescent light upon hybridization and illumination with visible light (see Figure 8-1).³⁴