Advanced Techniques in Diagnostic Microbiology

14. Signal Amplification Techniques 229
alkaline phosphatase (AP) to generate chemiluminescence. The amplified signal
on the target molecules is related to the number of target molecules. The signal
amplification is linear. Thus, the standard curve in each assay allows calculation of
the number of targets in the samples and therefore bDNA is a quantitative technology
and is used in the determination of viral load (Cao, 1995; Kern, 1996; Collins,
1997).
In general, there are seven steps of the assay which can be completed in 2 days.
The first two steps can be done on day 1 and the rest on day 2. The first step in the
assay is to release the nucleic acid from the target, such as virus, and is called the
target nucleic acid release. The release occurs through viral lysis buffer to disrupt
the virus, degrade nucleases (RNases), and release viral target RNA or DNA (DNA
targets require additional denaturation to yield single-stranded target). A detergent
such as proteinase K disrupts the viral coat to release the nucleic acid from the
virus and also inactivates RNases.
The released nucleic acid is captured to a solid surface by multiple capture
probes either in a microwell plate or in solution. The second step is target probe
hybridization and capture, or so-called target capture. Capture probes hybridize target
nucleic acid to the capture probe–coated microwell, and target probes hybridize
to the target. The oligonucleotides called capture probes (in solution) hybridize to
multiple sites on the target viral RNA as well as the capture probes that are coated
on the microwells. The target probes also hybridize to multiple sites on the target.
They will hybridize the next oligonucleotide added to the samples. The target viral
RNA is thus “captured” to the microwell through the hybridization of the two types
of capture probes in solution and on the microwell.
The next step, called preamplification probe hybridization (to target probes and
thus to the microwell), can be performed on the second day. After the overnight
incubation, the microwells are washed to remove unbound capture probes, target
probes, lysis reagent, and cellular debris. Preamplifier probes are added to the
microwells. Each preamplifier probe hybridizes to two adjacent target probes in
a cruciform configuration or cruciform design. One leader binds target probes
at 5 ′ end. There are 14 preamplifier sites with 7 linker sites for ligation. After
preamplification is the amplifier probe hybridization. Amplifier probes are added
to the microwells. They then hybridize and bind to preamplifiers. There are multiple
amplifier-binding sites present on each preamplifier for the amplifier probe
to hybridize to the preamplifier and form a bDNA complex or so-called signal
amplification multimer for amplification. Thus, the amplifier molecule is the key
to bDNA technology (Horn and Ureda, 1989).
The next step is the alkaline phosphatase (AP) labeled probe hybridization.
AP-conjugated probes called label probes are added to the microwells and hybridize
to immobilized amplifier complex. There are multiple label probe-binding
sites present on each amplifier. Dioxetane substrate (Lumi-Phos Plus, Lumigen,
Detroit, MI, USA) is added to the microwell for signal generation. The dioxetane
substrate chemically reacts with the alkaline phosphatase from the label probes,
which excites an electron, resulting in emission of a photon of light producing
chemiluminescence (Beck, 1990).