GBI – Genera Biosystems Ltd.

Advanced Molecular Diagnostic Systems – Multiplex qPCR


AmpaSand beads are silica microspheres that have been extensively modified using Genera’s proprietary technology to create identifiable groups of beads that can be distinguished on a conventional flow cytometer (a standard piece of laboratory equipment). The beads act as a microscale platform on which a variety of biochemical reactions can take place, such as the binding of disease markers.

The ability to identify individual groups of beads within a larger group facilitates “multiplexing” of tests. Multiplexing is the term used to describe testing for several different markers simultaneously. This can provide more information and increase testing efficiency. For example, several different sexually transmitted diseases (STDs), such as HPV, chlamydia, and gonorrhea, can be tested with a single multiplexed test, rather than three separate tests. More generally, it opens up opportunities for Genera to develop a range of sophisticated diagnostic products.

AmpaSand bead systems offer advantages over latex bead technologies. Silica beads are stable at 1000 ° C, and this thermal stability allows certain processes, some of them proprietary to Genera, that would not be possible with latex beads (e.g. high stringency DNA hybridization and PCR / hybridization processing single tube).

Solid-phase PCR target amplification uses specific primers (“Probe” primers) conjugated to AmpaSand Beads and red fluorescence-labeled aqueous (reverse) primers (see figure below). Additional unlabeled (forward) aqueous primers are included in limiting concentrations to enrich the target and promote solid-phase PCR. As a consequence of the solid-phase amplification process, the red reverse primers are incorporated into the growing PCR product and thus increase the intensity of the red color associated with AmpaSand Bead.

After PCR, the beads are analyzed on a flow cytometer, allowing differentiation by size and intensity of the green color of all target-defined bead populations. A significant change in the red fluorescence of a population of beads is indicative of a successful solid-phase PCR amplification event. Positive “calls” for target pathogens occur when the median red fluorescence of a population of beads exceeds its experimentally determined threshold level.

QPlots, Genera Biosystems’ proprietary software, uses these thresholds, as well as data from the internal calibration controls on each test plate, to make objective calls for each pathogen for each clinical sample. Results from QPlots are stored electronically and printed in an easy-to-read format.

The PCR reaction is essentially a nested PCR containing AmpaSand Beads with an immobilized internal forward primer. This primer contains a linker region that allows covalent attachment to the bead, as well as the active amine moiety for attachment of a fluorescent tag. Genera uses a green fluorescent label for bead marking on its current diagnostic kits. The liquid phase forward primer and fluorescence-labeled reverse primers act as external primers.

As PCR progresses, the forward strand grows from the immobilized primer (shown as a black dotted strand), and the fluorescently labeled reverse primer is incorporated into the complementary strand (red dashed strand). This strand gives the bead the color for the determination of positive or negative: the more complement, the brighter red the bead and a positive call. If there is no target in the sample, there is no amplification and therefore no color change in the bead.