New Generation Testing Platforms

The latest generation of genetic detection technology has recently become available and is already used in industries where specificity is critical. These systems use probe-based technology to replace the melt-curve analysis. Probes provide additional specificity other than provided by primers. Because the probe’s structure includes a second highly specific single-stranded DNA sequence complementary to the target DNA, it will bond directly to the target DNA and produce a fluorescent signal that can be detected by an instrument at the time that the binding occurs. In contrast to non-specific dyes like SYBR Green, probes only bind and emit signals in the presence of the target DNA.

In the absence of target DNA, the fluorescent molecule or fluorophore is held in close proximity to a quencher molecule. This suppresses the fluorophore’s signal and no fluorescence is generated.

However, if target DNA is present, the probe’s complementary DNA sequence will bind to the target causing the probe to become linear. This spatial separation of the quencher and fluorophore generates the fluorescent signal. The amount of signal emitted is directly related to the quantity of target DNA present in the reaction tube. With each PCR cycle, the target DNA is copied and the signal increases, thus eliminating the need for the post amplification melt-curve analysis.

Advances in Sample Preparation

Equally significant have been advances in sample preparation procedures and instrumentation-the other two components of a DNA-based detection system. Systems have been developed that allow for an upfront immunomagnetic separation (IMS) step to concentrate the target organism from a sample broth.

One newly developed procedure uses antibody coated beads and a novel concentration device to facilitate the IMS procedure. The PCR process outlined above assumes that the process starts with clean DNA; however many food samples contain materials that can inhibit or interfere with the PCR process.

The traditional approach used by PCR systems to eliminate these inhibitors is to dilute the sample after enrichment to decrease the concentration of inhibitory compounds. The inherent drawback to this approach is a decrease in sensitivity as dilution reduces the concentration of the target organism as well as the concentration of inhibitors. Hence, longer enrichments times have been needed to arrive at adequate levels of target organisms to compensate for dilution.

In contrast, using IMS to capture and concentrate the target organisms prior to the PCR procedure rather than dilution, allows for a shorter enrichment time as fewer organisms are needed at the end of enrichment. Additionally, the captured organisms are physically separated from the food matrix and enrichment media, leaving behind potential PCR inhibitors without decreasing sensitivity.

Instrumentation Advances

A significant advancement in instrumentation has been the introduction of thermocyclers with incorporated multichannel detectors. These instruments can read multiple fluorescent signals simultaneously, allowing for multiple targets to be read within a single amplification tube. More importantly, this technology eliminates the need for melt curve analysis required by single channel instruments which rely on the melt curve and non-specific indicator dyes to differentiate between amplified DNA segments. Multichannel instruments allow results to be determined with each cycle, providing real time results.

Additionally, rotary format thermocyclers add further accuracy and speed to a genetic detection system. Conventional thermocyclers use a Peltier block format that requires heating and cooling of a rectangular shaped block containing the amplification tubes. The thermal process utilizing a block format is less efficient as heat must be transferred through a solid service. This is a slow process, and also can create cold spots around the perimeter and in the corners of the block. To compensate for this, block cyclers require longer hold times during each cycle to allow the temperature across all parts of the block to equilibrate.