Improving PCR based detection methods

The polymerase chain reaction, commonly known as PCR, is arguably the most important technique in modern genetics. It is also the favoured method for detecting and quantifying GMOs. PCR holds a lot of potential for helping scientists solve new challenges with GMO testing. The technique, however, is costly, highly technical, time consuming, and often wavers in accuracy and precision.

Gel electrophoresis is a technique used to look at DNA. If a PCR test is positive, a dark band is visible on the gel. No band means the target DNA sequence is absent.
Gel electrophoresis is a technique used to look at DNA. If a PCR test is positive, a dark band is visible on the gel. No band means the target DNA sequence is absent.
PCR enables the detection of specific strands of DNA by making millions of copies of a target genetic sequence. The target sequence is essentially photocopied at an exponential rate, and simple visualisation techniques can make the millions of copies easy to see. If the targeted genetic sequence is unique to a certain GMO, a positive PCR test tells you that the GMO is present in your sample.

The method works by pairing the targeted genetic sequence with custom designed complimentary bits of DNA called primers. In the presence of the target sequence, the primers match with it and trigger a chain reaction. DNA replication enzymes use the primers as docking points and start doubling the target sequences. The process is repeated over and over again by sequential heating and cooling until doubling and redoubling has multiplied the target sequence several million-fold. The millions of identical fragments are then purified in a slab of gel, dyed, and can be seen with UV light.

Bringing PCR out of the lab

PCR tests require sophisticated equipment and expensive ingredients. Samples that need testing have to be shipped to GMO detection labs, which makes GMO testing slow and expensive. Co-Extra researchers are currently devising ways to bring PCR capabilities on-site – to farms, ports, and cargo yards.

Enhancing the performance of quantitative PCR

If you’re trying to find out if a GMO is present in a sample, traditional PCR can easily give you a yes or no answer. In other words, PCR is essentially a qualitative test. European legislation on labelling and traceability, however, tolerates a low level of accidental mixing. This makes a simple yes or no insufficient for determing if a product needs to be labelled. Finding out the exact percentage of GM content in a sample has only been possible since the development of a technique known as quantitative real-time PCR. Quantitative real-time PCR gives a good idea of how much of the target sequence was present in a sample at the start of the PCR reaction.

Quantitative real-time PCR machines track how quickly the copying of the target sequence reaches an exponential rate. The more GMOs in the sample, the sooner the target sequence reaches an exponential rate of amplification.

A computer attached to a quantitative real-time PCR machine logs the rate of amplification and produces images with a rate curve for each of the samples. To make a concrete reading from an unknown sample, the unknown test sample is compared to standards with known percentages of GMOs. For example, if the reading for the unknown sample falls between the readings for the 0.7 percent and 0.8 percent references, you can be quite confident that the GM content of your test sample lies between 0.7 and 0.8 percent.


It's all relative - Alternative reference material for quantitative PCR

Trying to carry out a quantitative GMO test without standard reference material is like trying to read a thermometer that hasn’t been calibrated (i.e. a thermometer without lines and numbers). Although you would see something on the screen, you wouldn’t be able to make any sense of what you’re seeing.

If a laboratory wishes to test a sample of maize grains for GM content, it will have to carefully prepare standard mixtures of GM and conventional maize grains to use as reference material. Any reading from quantitative PCR for an unknown sample can only be interpreted when it's looked at relative to known references.

To make reference material, laboratories grind grains and mix them at set proportions such as 0.1percent GM, 0.5 percent GM, 1percent GM, and so on. Nowadays it’s possible to order pre-made, certified reference material, but it is not yet available for all GMOs or for every matrix (i.e. form of the material e.g. grains or starch).

Preparing standards is very time consuming, and even certified reference materials can be a source of uncertainty. For these reasons, scientists have been considering new kinds of reference material. Rather than extracting DNA from reference material in the form of ground seed mixtures, a step could be removed by starting with purified DNA itself. Reference material could be produced by carefully mixing pure, synthetic DNA identical to the targeted genetic sequences for the GMO and the derivative, conventional crop. This would circumvent a major step in preparing reference material, increasing efficiency and making tests more reproducible.


The modular approach to GMO testing

Quantitative PCR is a sensitive technique that can give precise results, but it is prone to variability based on many factors relating to personal technique and materials. Before any given quantitative GMO testing protocol can be considered official, it must have undergone a validation process specific to the reference materials, the specific GMO, and the matrix being tested. For instance, different test protocols must be validated for testing the Bt maize MON810 and for the Bt maize T25. Likewise, separate protocols exist for testing maize kernels for MON810 as opposed to testing a tortilla. Not only is this complicated, it makes validating tests for every possible matrix and for each individual transgenic event virtually impossible. The problem will only become more complex as more and more GMOs are developed and commercialised. This is why researchers are working on what is known as a modular approach.

The modular approach takes GMO tests and validates them step by step. For example, GMO testing involves sampling followed by preparing the material for use in the lab, then extracting DNA, determining how much of the plant under investigation is present, and finally determining how much of that is GM. Although certain steps will be GMO- or matrix-specific, some steps will be common to many different tests. In the modular approach, common steps are validated separately so that they do not need to be validated again and again. These standardised “modules” can be carried over and used with new test matrices and new GMOs.


Killing two birds with one stone

PCR requires specialised equipment and expensive reagents. More efficient, cost effective PCR-based tests are needed to make routine testing economically feasible. Using current techniques, PCR generally tests for only one GMO at a time. Maize alone has more than a dozen authorised transgenic lines in the EU, with many more already authorised in other countries. Research is now underway to develop multiplex PCR methods that can simultaneously detect many different transgenic lines.


Single GMOs with more than one GM trait

Another major challenge is the increasing prevalence of transgenic crops with stacked traits. This refers to transgenic cultivars derived from crosses between transgenic parent lines, combining the transgenic traits of both parents. One GM maize variety now awaiting a decision by the European Commission, MON863 x MON810 x NK603, has three stacked traits. It is resistant to an herbicide and to two different kinds of insect pests. Some combined testing methods could give results that would triple the actual GM content of a sample containing this GMO.