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From: Food Quality & Safety magazine, June/July 2005

New Generation Testing Platforms

DNA-based systems have continually evolved for successfully detecting pathogens.

by David E. Kerr

In the last few years, technological advancements of DNA detection systems, more commonly known as polymerase chain reaction (PCR) technology, have been remarkable. In fact, parallels can be drawn to the computer industry, where technology that was cutting edge as little as four years ago, barely meets our most basic expectations today.

The newest generation of PCR systems offers several important benefits to food manufacturers, namely unprecedented accuracy and in some cases, faster time-to-results. With the challenges imposed by test and hold programs, these advantages can have a large impact on a company's bottom line and provide them the necessary edge needed to succeed in a highly competitive marketplace.

Basics of Genetic Detection

To better understand the latest technology advances, it is critical to understand the principals of genetic detection. Since PCR occurs at the molecular level, identifying and copying specific segments of DNA unique to the target organism, it provides a greater degree of inherent specificity compared to the antibody or antigen-based detection systems.

To begin the PCR process, the target sequence of DNA is identified by a primer -- a single strand of DNA with a sequence complementary to a portion of the target DNA. Once identified, the target DNA is quickly replicated or amplified.

Amplification is an exponential process whereby one copy becomes two, then four, etc., until, after a short period of time, millions of copies of the unique DNA sequence have been created. As a result, PCR has the ability to detect the presence of just a few target organisms much faster than other methods. This entire process of identifying and copying DNA segments is accomplished by placing a sample through a repetitive series of precisely-controlled temperature changes, a procedure know as thermal cycling.

PCR Generations: Then and Now

Early versions of PCR employed a fairly simplistic amplification procedure relying on primers alone for specificity. Reading and interpreting results required the use of electrophoresis gels, which were susceptible to contamination from adjacent samples and the environment. Gels were also difficult to interpret and the process was time-intensive and technique-dependent. This technology was used primarily for research, and due to its limitations, was never broadly adopted by the food industry.

The second generation of PCR systems, introduced in the late 1990s and still widely used, represented some significant improvements to this technology.

For the first time the entire PCR reaction, including detection, occurred inside of a PCR tube, eliminating the need for electrophoresis gels. Usability was greatly enhanced which allowed for the adoption of this technology by more companies within the food industry. In place of gels, detection was now performed by using a non-specific DNA binding fluorescent dye, such as SYBR Green. This dye attaches to all double stranded DNA found in a sample. When bound to DNA, the SYBR Green emits a fluorescent signal. The intensity of this signal is directly related to the amount of double-stranded DNA. This unique property of the dye can be used to indicate the presence of the target DNA through a process known as a melt-curve analysis. After the DNA amplification process has been completed, the sample is subjected to a gradual temperature increase, which causes the amplified DNA to dissociate and release the SYBR Green dye. Because the DNA and the dye will separate across a narrow temperature range, careful analysis of the associated drops in fluorescence may reveal the presence of the target DNA.

While easier to use, the non-specific binding properties of the dye lead to the production of multiple melt curves, which can make interpretation difficult and may limit the specificity of the assay. Additionally, since the melt-curve analysis must be performed after the DNA amplification step, it adds a significant amount of time to the assay (up to 2 hours).

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.

The rotary format cycler utilizes a centrifugal design to provide greater thermal efficiency. The amplification tubes are placed in a circular rotor and spun at high speeds, while heated air is directed from all sides. This allows for a higher degree of temperature uniformity and eliminates the need for the long dwell times required of block systems.

The advantages in speed and specificity that the next generation of DNA detection systems offers the food industry are significant, particularly in light of test and hold policies. The laboratory and operational costs of inaccurate test results can represent a significant financial burden to companies tying product release to test results. Companies conducting their own pathogen screening and those who send their sample to an independent lab for screening can benefit from the superior accuracy of this newest generation of testing platform.

Dr. David E. Kerr is director of pathogen detection technologies at BioControl Systems Inc. (Bellevue, Wash.). He can be reached at dkerr@biocontrolsys.com.

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