The most popular molecular biology technique used in our laboratory is the polymerase chain reaction (PCR). The majority of the plant viruses we work with have genetic material in the form of RNA, hence the method is often preceded by a reverse transcription reaction that enables us to obtain cDNA from the isolated RNA. The PCR reaction consists of a repeated cycle of three steps taken at different temperatures: denaturation, primer annealing, and DNA strand synthesis. The reaction takes place in a thermocycler. After the reaction, electrophoretic separation of the resulting products on an agarose gel by mass size marker is necessary. In the PCR reaction, it is essential to select appropriate primers for detecting the widest possible range of virus isolates, which are often genetically diverse. No less important is reaction specificity: the primers should be designed to amplify solely a specific gene or region in the pest genome.

One type of PCR is quantitative PCR (qPCR). The technique relies on fluorescent dyes or probes to monitor reaction product quantities during the reaction to eliminate the electrophoresis step after the reaction ends. TaqMan probes, which are short oligonucleotides with a fluorescent tag at the 5' end and its quencher at the 3' end, increase reaction sensitivity and specificity. Some qPCR thermocyclers offer the additional option of performing high resolution melt analysis of DNA after the reaction. This technique supports the detection of mutations, polymorphisms, and minor deletions and insertions. This allows for the quick detection of e.g. virus pathotypes that differ by single mutation in a given genome region.

Isothermal nucleic acid amplification by loop-mediated isothermal AMPlification (LAMP) has gained popularity of late. This technique involves a specific synthesis of new DNA strands on the template in subsequent reaction cycles and replacing DNA strands with newly synthesized equivalents. The reaction takes place at a constant temperature (commonly at 60-65°C). Moreover, the addition of appropriate fluorescent dyes to the reaction changes its color from orange to green under UV light where the result is positive and a pathogen is detected. Therefore the method does not require visualizing reaction products on an agarose gel. LAMP is not only rapid (a mere 15 minutes) in pathogen detection, but also highly sensitive. The use of 3 primer pairs increases its specificity. As no specialized equipment is required, the technique can be used to detect pathogens in field and greenhouse settings.