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.
Explore this issueJune/July 2005
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).