The PCR-DEIA format offers a high-throughput platform for rapid hybridization of amplified DNA.
The test procedure comprises three parts:
Isolation of the DNA from the sample (reagents NOT provided in the kit)
Amplification of target DNA using PCR
Detection of the biotinylated product in our DEIA assay.
Double-stranded DNA products are generated during PCR whereby one of the primers is labeled with biotin. After amplification, the biotinylated PCR products are captured onto streptavidin-coated microtiter plate wells. Double-stranded DNA is denatured under alkaline conditions and the uncaptured (i.e. unbiotinylated) strand is removed by washing. A labeled oligonucleotide probe is added, which can specifically hybridize to the captured strand. Also, cocktails of multiple probes can be used. After stringent washing, hybrids can be detected by conjugate and substrate followed by optical density measurement.
An advantage of this method is the high throughput of the microtiter plate format and the availability of automates and equipment to reduce hands-on time. Therefore, the assay is very suitable to quickly distinguish between positive and negative samples as a first screening step in molecular diagnosis.
DEIA assays have the following advantages:
Hybridization is specific and multiple probes can be used in a single well for broad-spectrum screening.
Detection is highly sensitive.
DEIA has a high throughput, and requires well-established standard 96-well microtiter plate equipment.
The read-out can be performed manually or automated
The test is fast (about 8 hours, including amplification)