Development of the first PVM TaqMan® primer set and a one-step real-time multiplex DiRT-PCR for the detection of PLRV, PVY, PVM, PVS, PVA and PVX in potato tuber sap

Abstract

Testing for potato viruses is globally very important to prevent a critical shortage of potato supply. In most countries, testing is obligated by law. In Germany, seed potatoes are monitored for six viruses: PLRV, PVY, PVM, PVA, PVX and PVS. They can cause up to 90% loss of potato tubers in the field. Common methods currently used for testing are ELISA and conventional real-time PCR, but both are very time-consuming, and the former needs a high capacity of green houses and human resources, the latter elaborate RNA extraction steps. Recently, we proposed a new method called real-time DiRT-PCR which enables us to test for PLRV, PVY and PVS along with an internal control in three duplex real-time PCR reactions directly on diluted tuber sap. In this study, we describe the first TaqMan® assay for PVM published so far and embed it into a multiplex system to detect the remaining viruses. We are now able to sensitively test for the presence of six viruses in two multiplex reactions using the real-time DiRT-PCR without RNA purification.

A rapidDNAextraction method for the detection ofClavibacter sepedonicusandRalstonia solanacearumin potato samples in a multiplexPCRtest

“…Furthermore, the Cq ‐values for QE extracts were similar to those obtained with the standard DNA extraction method in place in the NPPO laboratory. The QE extraction protocol thus seems also suitable for RNA preparation and could possibly also be introduced into potato test schemes for virus diseases that usually have to cope with even higher sample numbers owing to the separation into subsamples (Prinz et al, 2022; Schumpp et al, 2021).…”

One-step multiplex DiRT-PCR for PLRV, PVY, PVM, PVS, PVA and PVX ready for routine testing directly on tuber sap

“…In Stammler (2020) and Prinz et al (2022) we developed a method, called multiplex real-time DiRT-PCR (= direct reverse transcription PCR). This method can detect all six important viruses plus controls in two quadruplex RT-qPCRs without prior RNA extraction.…”

“…This is possible by using the KAPA3G™ Plant PCR kit (former KAPA Biosystems, USA; now Roche, Switzerland, Schori et al, 2013;Stammler, 2020) and adding a robust reverse transcriptase, i.e. SIII (Merck -Sigma Aldrich, Germany) as described in Stammler et al, 2014, 2018, Stammler, 2020 as described in Prinz et al, 2022. So far, we validated this new method comparing single tuber heels to ELISA on leaves coming from the same tuber.…”

“…So far, we validated this new method comparing single tuber heels to ELISA on leaves coming from the same tuber. The agreement between methods ranged from the best rating "almost perfect" to the third best rating "fair" according to Gwet's AC1 agreement index, depending on the type of virus, being the best for PLRV and the worst for PVM (Prinz et al, 2022). However, single tuber testing (heel end) is not practical for routine testing and comparison to ELISA on leaves not ideal as it is not the same part of the plant and virus might be enriched unproportionally in the leaf (Stammler et al, 2018;Stare et al, 2015) or unequally distributed within the tuber (Dupuis, 2017).…”

“…However, single tuber testing (heel end) is not practical for routine testing and comparison to ELISA on leaves not ideal as it is not the same part of the plant and virus might be enriched unproportionally in the leaf (Stammler et al, 2018;Stare et al, 2015) or unequally distributed within the tuber (Dupuis, 2017). Hence, in the following study, we decided to use the protocol of the multiplex real-time DiRT-PCR as in Prinz et al (2022), improve the methodprotocol further for pool testing, (in short DiRT) and compare it to the results of ELISA on leaves of the same tubers and the commercial RE-kit on the same heel ends.…”