Technology

DNA extraction

Myconostica will utilise proven existing technology to optimise DNA release from fungi and other pathogens.
This is a critical step in success, and one that is in active testing currently.

Molecular targets

Myconostica has several proprietary molecular targets which it will utilise for its series of tests. Criteria for target selection are rigorous and the targets have been extensively tested.

PCR (or polymerase chain reaction)

PCR has been a key enabling technology in the growth of molecular diagnostics particularly in the area of virology. However, limitations in its use for pathogen detection have been apparent with relatively high levels of false positive results. PCR assays typically analyse amplicons at the end point of amplification and this has been shown by experiment to display poor precision, low sensitivity and limited dynamic range. Attempts to correct this with the use of post amplification analysis of the amplicons with probes has been partially successful but is technically demanding and hence the application to routine analysis has been limited.

Myconostica’s Real-Time PCR Chemistry

Molecular Beacon technology relies on DNA amplification using the well-established Polymerase Chain Reaction (PCR) system, with major enhancements. Total DNA is extracted from clinical samples and combined with a molecular beacon designed to detect one or more fungal species. As each original DNA sequence is specific, so amplification is specific.

If one or more copies of fungal DNA is present it will be amplified millions of times in ~90 minutes, providing great sensitivity and speed. The number of copies is monitored by the emission of light from individual beacon molecules during the process, and the time it takes to generate a detectable product is proportional to the amount present at
the beginning.

Each piece of target DNA (original plus amplified) binds to a beacon, allowing the fluorescent dye to be uncoupled from its ‘quencher', emitting light.

Real-Time PCR

The advent of Real-Time PCR is set to be the answer to many of these limitations. The fundamental aspect of all Real-Time PCR assays is the ability to amplify a target sequence and to assess the presence of target sequence in Real-Time within the amplicons produced, all within a sealed tube, thereby removing the need for separate-protocol post amplification analysis to obtain increased sensitivity. The sealed tube aspect is important in clinical laboratories as it decreases the risk of contamination considerably and the nature of the assay always makes the risk of amplifying contaminating DNA a risk.

The amplicons generated can be interrogated for the presence of defined sequence using self-reporting fluorescent probes with very high specificity, down to the level of single base changes. Signal is generated with these probes only if target sequence is present and this, in theory, allows for the presence of several target sequences to be determined simultaneously in so-called multiplex assays.

Figure 1

There are two major families of reporting probe molecules used in Real-Time PCR assays, linear probes and stem
and loop probes.

Molecular Beacons

Myconostica’s multiplex Real-Time PCR assays use the stem and loop Molecular Beacons as reporting probe molecules. Myconostica has licensed the use of Molecular Beacons from the Public Health Research Institute, PHRI, of Newark, New Jersey and works with the PHRI in the design of all its probes. See www.molecular-beacons.org

Molecular Beacons are single stranded oligonucleotide hybridisation probes of varying length. They are configured so that the probe sequence for the target DNA sits in the middle of the oligonucleotide. Each end of the oligonucleotide contains complementary sequences and in solution these sequences anneal to form a double stranded ‘stem’ with the probe sequence, remaining single stranded, as a loop structure (See figure below). One end of the oligonucleotide has a fluorescent reporter molecule covalently attached and the other end has a quencher molecule. The double stranded stem structure holds the quencher in close proximity to the reporter with the result that Molecular Beacons do not fluoresce in their stem and loop structure.

Figure 2

Molecular Beacons function in Real-Time assays by their probe sequence annealing to the target DNA. Molecular Beacons are configured such that the formation of the probe target hybrid is more energetically favourable that the stem hybrid. Binding of the Molecular Beacon to the target sequence causes the probe to assume a linear structure with a resultant significant increase in the spatial distance between the reporter and the quencher. Thus, when annealed to target DNA, Molecular Beacons fluoresce. (See Figure 2)

Molecular Beacons are capable of being designed to provide very high sequence discrimination if the binding energy of the probe-target Hybrid is only more favourable than the stem hybrid when a perfect match between probe and target sequence exists. This sensitivity is routinely used to determine single nucleotide polymorphisms in targets. Molecular beacons are clearly the gold standard real-time detection platform, combining incredible sensitivity and specificity with the ability to combine many different tests together by multiplexing.

Probe design

Probe design starts with selecting the probe sequence and determining the theoretical melt temperature of the
probe-target hybrid. Several computer programmes are available to determine this. It is important that under the PCR annealing conditions, the probe will bind to the target. After probe design, the two complementary arms on both sides of the probe sequence are designed such that they retain their structure at the annealing temperature of the PCR reaction in the absence of target. In the presence of target a probe target hybrid is the most energetically favourable and the event is reported as the Molecular Beacon assumes a linear configuration. Finally, at the chain extension phase temperature, the probe sequence is designed such that the probe target hybrid melts resulting in no probe degradation from the Taq Polymnerase. After melting, the Molecular Beacon reassumes the loop and stem structure and becomes dark. Obviously, this development work has been done in Myconostica’s kits.

Multiplex assays

If different fluorescent reporter molecules are used, several different Molecular Beacons may be used in the same assay allowing multiple targets or polymorphic variants to be detected in the same multiplex assay. Molecular Beacons are ideally suited to multiplex assays due to their high discriminatory power coupled to their low
background fluorescence. The low background fluorescence in Molecular Beacons is a function of the non-FRET quenching mechanism: The proximity of the quencher to the reporter allows both molecules to share electrons and thereby remain dark. Fluorescent energy is released as heat. The same quencher can be used for a range of reporter molecules making Molecular Beacon design relatively straightforward, even when multiple reporter molecules are used. This is in contrast to FRET based dye pairs where the constraint of overlapping spectral characteristics and incomplete quenching make assay design more problematic.

Furthermore, Molecular Beacons are not degraded during the Real-Time PCR reaction and therefore do not contribute to an increase in background signal. Molecular Beacons are designed for an assay so that fluorescence
is only obtained when exact target sequence is present. Due to their mode of action, Molecular Beacons are characterised by a low noise to signal ratio, which allows minority target sequences in a sample to be detected even in the presence of significantly larger amounts of other target sequence.

Molecular Beacons have additional features making them attractive for diagnostic assays: They are capable
of being synthesised with high quality; they are relatively easy to optimise to the degree of sensitivity the assay demands; they are not destroyed during the PCR and probe target binding may be confirmed by melting analysis. Furthermore, Molecular Beacons have been included in assays that have attained regulatory approval from the FDA.

Myconostica envisages many of its diagnostic products will be multiplex assays and for this reason has chosen to use Molecular Beacons in its assays.

Real-Time Platform

There are numerous real-time thermocyclers on the market currently (summary of machines available here)
and Myconostica will provide tests for several. Detailed run conditions will be provided for each machine, together with detailed interpretation guidelines.

Controls

Myconostica has a suite of appropriate and simple controls to ensure that the results are robust and clinically reliable.

Interpretation

Detailed guidelines are provided with each test to ensure that the results can be reported to clinicians with confidence. In the first instance quantitative results will be used, but as confidence with the tests grows,
quantification may become useful clinically.