Plant Health Protection and PCR

In terms of plant health protection, there are many factors that can influence the occurrence of disease in commercially and socially valuable plants. Both population growth, which favours monocultures, and a change in climate, resulting in expansion of pathogen and weed species ranges, can put pressure on a system that provides an ecosystem service to humans and other species.

Wherever you look you can see where plants have been used, whether it is for the furniture in your sitting room, the food on your dining table, or in the environment of your favourite walking location. It is therefore necessary to follow guidelines on ensuring pathogens and competitive species are minimised, and that research is used to improve resistance against these agents. Current European Union regulations, such as Council Directive 2000/29/EC, aim to minimise the introduction and spread of organisms that are harmful to crops, fruits, vegetables, cut flowers, ornamentals, and forests. This is aided by plant health protection and the maintenance of public and private green spaces, forest, and ‘natural’ areas. Other measures include the use of plants of known provenance, the restriction of imports from outside the EU, the regulation of movement, and the enforcement of containment or eradication of materials and plants infected with pathogens or harmful species. It is easy to see that the impact of unwanted organisms can have an impact that is both financially detrimental and socially damaging. Recently we have had several examples of this happening. Ash dieback, Hymenoscyphus fraxineus is a fine example of where a disease has posed a threat to ornamental stock. Meanwhile, in agricultural settings, black-grass, Alopecurus myosuroides, can reduce viable yields due to crops being out-competed in terms of space, nutrients, and water.

Various technological advances have been made in recent years which includes innovations such as pregnancy style tests, like the Black-grass Resistance Diagnostic tool, developed by Newcastle University and Mologic Ltd, which detects glutathione transferase AmGSTF1 that occurs in high concentrations in plants that are herbicide resistant so that farmers can have an accurate idea of the effectiveness of their treatments, and make necessary changes to save both crops and money. Another piece of technology that is relatively new is a spore sampling unit, developed by Rothamsted Research, that works on-site in the field, providing a rapid real-time overview of pathogens (bacteria, fungi, and viruses) in the air.

“the fundamental technique that compliments the development of these innovative early detection tools is DNA analysis”

The fundamental technique that compliments the development of these innovative early detection tools is DNA analysis. A quantitative real-time Polymerase Chain Reaction (qPCR) technique is often used to provide rapid, sensitive, and accurate detection and quantification of pathogens, discrimination between species and sub-species of viruses, bacteria, fungi, oomycetes, and also for detecting specific genetic sequences that are present in pathogens and weed species, by amplifying around 100 base pairs in real-time by using fluorescence and quantifying PCR products. To this end it is possible to undertake research in the biology in a very accurate and efficient way.

When choosing a system, it is important to choose one that is efficient, rapid, sensitive, and has good performance and reproducibility. The PCRmax® Eco 48 fulfils these aspects and is ideal for a range of uses, including: academic, government and corporate laboratory research. The PCRmax Eco 48 real-time PCR system is a MIQE compliant, high specification, multiplexing, economically priced real-time thermal cycler that accommodates a unique 48-well polypropylene PCR plate utilising the same geometry as standard 384-well plates, but only 1/8 of the size. This enables users to dramatically reduce the qPCR reagent volumes compared to traditional 96-well instruments, saving users precious sample, whilst still producing a strong fluorescence signal. A minimum volume of 5μl is validated, resulting in a more efficient use of expensive and hard to acquire template DNA samples. Minimising the plate size also significantly improves thermal uniformity.

Fast, uniform temperature control is important because accurate dwell temperatures ensure primers bind most efficiently and polymerase enzymes work optimally, generating the maximum yield of target DNA, thus ensuring specificity and efficiency. The unique thermal block design of Eco 48 achieves this with a unique heating and cooling system that provides accurate ±0.1ºC temperature control and quickly cycles from one temperature to the next. The Eco 48 consists of a precisely electroformed 48-well hollow silver block containing conductive fluid, whilst the block is heated and cooled by a single Peltier device, with an agitator assembly consisting of two paddles driven by electromagnetic motors.

This design allows fluid to rapidly circulate across 48 wells and enables high ramp rates and reduced experimental time and virtually eliminates thermal non-uniformity and prevents edge effect, resulting in higher qPCR performance, tighter Cq, greater PCR efficiency, higher R2 and the ability to perform demanding HRM applications. Eco 48 thermal control is superior to traditional 96-well peltier systems where thermal efficiency decreases as distance from the peltier increases. Eco 48 has a function in the software where different experiments are reliably combined so that well-to-well, plate-to-plate and instrument-to-instrument results are as consistent as running a single experiment.

In addition to uniform temperature control the Eco 48 delivers precise and sensitive fluorescence detection, facilitating all 4 colour multiplex applications due to a high-performance optical system. The system works by speeding up data collection because only 1 image is required to cover the full sample dynamic range. It reduces the influence of a highly fluorescing sample on adjacent wells and avoids ‘blooming’, whilst maximising sensitivity by permitting an appropriate exposure level for each well, by dye.

With Plant Health Protection being linked to economic challenges, it is important to develop methods of monitoring plant health. This often involves genetic research which is aided through the use of efficient and highly uniform PCR systems. The Eco 48 is an ideal system for this application.