PhytAlert Turf and qPCR technique: key for turf disease

For decades, the qPCR technique has been a widely used and indispensable technique for both medical and biological laboratories. Today it is popularly known worldwide, the detection technique for COVID-19 and is the key to a healthy turf, a novel way to study turf, which Tiloom introduces to the turf industry with the innovative kit PhytAlert Turf from Microgaia Biotech.

New ways of studying the health of our turfgrasses are opening up thanks to qPCR.

New ways of studying turfgrass health are opening up around the world thanks to technology put into practice with our new Phytfieldlab kit.

This technique is based on the exponential amplification of DNA molecules, using the enzyme thermostable DNA polymerase. Its acronym is PCR: Polymerase Chain Reaction. It is now an accessible technique so that all fields can be diagnosed quickly and accurately.

We can now apply this technique to know the phytosanitary status of our plants. sports surfacesIn other words, we can identify the pathogenic micro-organisms, both in the plant and in the soil, before they produce the disease. This allows the greenkeeper to make decisions about the type of treatment to use, assessing the real need for its application, saving costs, optimising treatments and reducing the presence of these chemical compounds in the soil.

With the KIT PhytAlert Turf you can diagnose grass quickly and accurately.

The main advantages are:

• Control and monitoring: allows to anticipate before the first phytopathogenic symptoms appear.
• Prevention: analysis of soil condition prior to establishment of turf or any crop.
• Certification of pathogen-free turf prior to installation on our stadiums or greens.
• Diagnosis of diseases in both symptomatic and asymptomatic lawns: targeted and effective treatments, avoiding the use of broad-spectrum products.
• Wide range detection (substrate, plant, water, seeds).
• Detection and quantification of micro-organisms before and after treatment with plant protection products to check their efficacy.
• Quality control of products formulated on the basis of beneficial micro-organisms.

Differences between a PCR and a qPCR

In the conventional PCR technique, if you want to check whether the DNA has amplified, and therefore whether the micro-organism of interest is present in the sample or not, it is necessary to carry out another additional technique called electrophoresis. However, even when performing electrophoresis and checking that the DNA has amplified, at no time is it possible to quantify the DNA that we have amplified, i.e. we cannot know whether we have a high or low quantity of the microorganism that we want to analyse.

There is a version of the PCR technique, called real-time PCR or qPCR, which allows, in addition to detecting the existence of these microorganisms early, the quantification of these microorganisms in real time, immediately. Thus, the qPCR technique has a clear advantage over conventional PCR, as it allows us to check the real level of infection by pathogenic microorganisms, and whether their presence in the lawn is relevant or not.

qPCR OPERATION

qPCR is a technique that allows the duplication of DNA from a small amount of this molecule, known as template DNA. This is made possible by an enzyme, DNA polymerase. This technique allows the amplification of the DNA to be monitored thanks to the signal produced by a fluorescent marker attached to one of the components of the reaction, which will provide a light signal of intensity proportional to the amount of DNA being produced. Thus, the more DNA there is, the more light the fluorescent marker will emit.

The emitted signal is intercepted by software that collects the readings in a curve representing the entire amplification reaction cycle.

This equipment is called Thermocycler, and as its name indicates, it is in charge of carrying out the temperature cycles necessary for the DNA amplification reaction to take place. The set temperature cycles are repeated about 40 times, and in each of them, the following steps take place:

• 1st part of the cycle, temperature 95º. The template DNA molecule is denatured.
• 2nd part of the cycle, temperature 60º. Binding of the primers to the template DNA molecule.
• 3rd part of the cycle, temperature 72º. DNA polymerase elongates the DNA strand from the primers that have been attached to the strand in the previous step by eluting dNTPs.

The highest diagnostic technology available for your field.

In each temperature cycle performed by the thermal cycler, a copy of each of the DNA strands is produced, so that if we start with a new DNA molecule, we will obtain 2 DNA strands in the 1st cycle, 4 DNA strands in the 2nd cycle, 8 DNA strands in the 3rd cycle and so on until the last cycle, where we will have obtained millions of DNA strands.

When DNA amplification occurs in a qPCR reaction, it is possible to see in real time how the DNA molecules are being duplicated. This is because one of the components of the reaction, the probe, is labelled with a fluorescent compound, which will emit light only when the DNA strands are duplicated. Thus, the more DNA strands are duplicated, the more light the marker will emit, which will be captured by the thermocycler's sensors and finally interpreted by the software.

At the moment when the light generated by the fluorescent compound starts to be significantly visible (indicated by a curve in the software), it means that enough copies of the template DNA have been made for the template DNA to be detected. This moment is known as the detection threshold, which means that from that cycle onwards, detection of the template DNA has occurred. In the case that the template DNA comes from a micro-organism, it can be said that detection of that micro-organism has occurred.

How do I interpret the information provided by a qPCR?

The information obtained from a qPCR reaction varies depending on the reason for performing the reaction.

In the case of detection of plant pathogenic micro-organisms, the information being sought is the detection of these pathogens by DNA. It is also extremely useful to know how much of the micro-organism is present in the sample analysed.

In the event that DNA of the microorganism sought is present, during the qPCR reaction, we can see in real time whether DNA duplication occurs thanks to the signal emitted by the fluorescent marker. An exponential amplification line appears at the detection threshold, which, as mentioned above, is the moment at which it can be said that the microorganism is present in the sample.

If the detection occurs in a short time, i.e. in early cycles, it means that we have more microorganisms than if the detection occurs in late cycles. This is based on the fact that if we start with a higher number of DNA copies, and therefore a higher number of microorganisms, we will reach the necessary DNA copies earlier for the thermal cycler sensors to detect them.

Tiloom together with Microgaia wants to technify the sports turf sector and by means of our KIT PhytAlert Turf you can go one step further in plant health.