Protocol overview
First, a portion of the enrichment culture is placed into a test tube. A lysis reagent is added, which disrupts the cell and releases the nucleic acid target molecules. A portion of the lysed sample is then transferred to a microwell and the probe reagents are added. (Figure 1.)

The probe reagents consist of: 1) an oligonucleotide capture probe specific to rRNA sequences of the target organism and labeled at the 3’ end with polydeoxyadenylic acid (poly dA); and 2) an oligonucleotide detector probe also specific to rRNA sequences of the target organism and labeled at the 5’ end with the enzyme horseradish peroxidase (HRP). The hybridization reaction is then allowed to proceed for one hour. If target rRNA is present in the sample, both probes will hybridize to their complementary sequences on the target molecule.

The resulting complex is captured onto the solid phase coated with polydeoxythymidylic acid (poly dT), which is complementary to the poly dA portion of the capture probe. Unbound probe is then washed away, and a substrate of HRP is added.

Following a short incubation, blue color indicates the presence of hybridized detector probe in the complex and thus the presence of rRNA from the target organism. Stop solution is added to convert blue to yellow color and results are determined spectrophotometrically at 450 nm. An absorbance value in excess of a predetermined threshold indicates a positive test result. (Figure 2.)

Figure 2.