Colombia: System detects up to 5 potato viruses at the same time

To test the development of his technique, which acts as an X-ray “scanner,” microbiologist Neider Alfonso Jiménez Miranda, Master in Microbiology from the National University of Colombia (UNAL), worked with 2 commercial varieties of potato: diacol papyrus and yema de huevo, and with 8 native varieties, including alcarrosa and balbanera, from the Boyacá municipalities of Ventaquemada and Chiscas.

The method applied by Master Jiménez combines techniques used separately in other research to detect several viruses at once, which is important since current tests only detect one, and are also difficult to access for small potato farmers.

For example, the proposed methodology would allow entities such as the Colombian Agricultural Institute (ICA) and the Colombian Agricultural Research Corporation (Agrosavia) to offer a more precise and focused service.

The proposal combines the Multiplex RT-PCR technique – which amplifies the genetic material of different viruses in a single reaction – with qPCR, a tool that not only confirms the presence of the virus in real time, but also measures how many copies there are in the sample, as if we were counting how many viruses there are in each plant.

The secret was in identifying specific primers, which work like small keys that only fit into the genetic material of each virus. In other words, if there is a virus in the sample, the primers find it and amplify it, allowing it to be detected.

To ensure that each key fit perfectly, Master Jimenez used bioinformatics analysis programs that optimized his design, ensuring that the test was accurate and did not produce false positives.

In a single test

For the study, 100 adult plants (2 months old) were analyzed in the laboratory, grown in flasks with agar, a substance that provides the necessary nutrients and allows them to grow in vitro, to take tissue samples and analyze the viruses; in addition, two multiple detection systems were implemented, which allowed 5 viruses and a viroid to be identified in about 3 hours, which reduces time and resources compared to doing it separately.

Some of the viruses detected were PVY (potyvirus), transmitted by aphids, which causes leaf death and tuber deformation; PVS (carlavirus), which is also transmitted by aphids or by contact between contaminated plants, and is generally asymptomatic, making it difficult to recognize; and the potato yellow vein virus, which is transmitted by whiteflies and turns the veins of leaves yellow. When these diseases appear or come together, losses can range from 10 to 80%, depending on when action is taken.

Finally, to check the results, the scientists used agarose gel electrophoresis, a technique that allows the genetic material of viruses to be “seen” in a kind of gelatin, in which, by applying an electric current, the changes specific to each virus can be observed in an ultraviolet light transilluminator, a machine that shows a “fingerprint” or band of each of these pathogens.

Thus, instead of analyzing each virus separately with the conventional PCR technique – famous during the Covid-19 pandemic – this method allows the detection of the presence of the virus and facilitates the amplification of certain areas of the plant genome to see the genes that are activated when infected.

This breakthrough not only allows viruses to be detected in crops more quickly and affordably, but also lays the groundwork for creating diagnostic kits, so that in the future, entities in charge of carrying out field tests will not need to invest in expensive laboratories; in addition, potato farmers could have seeds that are completely certified as virus-free, guaranteeing better production.

The work of Master Jiménez was directed by professors Fabio Aristizábal, from UNAL, and Zaida Ojeda, from the Pedagogical and Technological University of Colombia.