Eppendorf Biochip Systems is developing a technology called RAP™ (Real-time Array PCR) or RAP-ID™ that combines the advantages of qPCR and microarrays, i.e., the multiplexing capabilities of microarrays with high sensitivity, wide dynamic range, and potential for quantitative results typical of real-time PCR.
Sven Bülow, Ph.D., managing director of the Hamburg, Germany-based company, labels the technology a “next-generation molecular diagnostics platform.” Eppendorf has filed 12 patent families on the methodology, which could be valuable for diagnostic applications having multiplex requirements in the range of about 10 to 100-plex and beyond, according to Dr. Bülow.
Dr. Bülow explains that while real-time PCR is the technology of choice for many molecular diagnostic assays, it suffers from a major hindrance: only a limited number of targets can be amplified and tested in a single tube or well at the same time despite the increasing need for multiplexing in molecular medicine.
“RAP is a single closed device that can produce results in about two hours or a maximum of three hours,” he says. “Initial applications for the technology include bacterial pathogen detection such as ventilator-assisted pneumonia and sepsis. Others include in vitro diagnostic applications in virology (multiple virus targets) and oncology, and food and feed testing (e.g., a biochip to detect GMOs in food).”
PCR Product Detector
The first assays that were successfully developed used the microarray as a detector for the PCR products. The amplicons resulting from the PCR were hybridized immediately onto the microarray, and the presence of the amplicons was verified by the signal of the corresponding capture probe spot. These assays are intended for the detection of bacterial species and antibiotic resistance.
“Another challenge was to perform multiplex amplification combined with real-time detection on the microarray,” says Dr. Bülow. The goal was to combine PCR amplification with array detection (hybridization) in a closed single device to eliminate contamination risks and offer an easy-to-use approach.
“The project was successful in developing a prototype instrument, detector, and consumable, as well as proprietary surface chemistry and detection modes. The first application will be the rapid detection of nosocomial pathogens and their antimicrobial resistance(s), which is a main healthcare issue worldwide, especially the need for speed and multiple data,” notes Dr. Bülow.