Development of the first PVM TaqMan\xae 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

Mirjam Prinz,Gerda Bauch,Johanna Stammler,Johannes Hadersdorfer,Adolf Kellermann

doi:10.1007/s10658-021-02436-z

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.

Highlights

Potato viruses can cause a major loss of yield and, are an important problem for seed potato production
For Potato Virus M (PVM) we found 78.8% general agreement with 65.2% of the samples being positive and 12.8% being negative, 15.3% were solely positive with double-antibody sandwich (DAS-)ELISA and 6.7% were solely positive with real-time DiRT-PCR
Assuming no falsepositive and false-negative results, the coverage of all PVM isolates of the used primer set and considering the extreme sensitivity of real-time DiRT-PCR in the sensitivity comparison of methods, we propose for PVM high natural viral replication frequency during virus enrichment phase of the growing-on DAS-ELISA protocol that filled the sensitivity gap obtained between real-time DiRT-PCR and DAS-ELISA to obtain high agreement between both protocols

Summary

Introduction

Potato viruses can cause a major loss of yield and, are an important problem for seed potato production. Potato plants can be infected by one or several viruses in two ways. Viruses are transmitted to host plant via aphids and spread to the tubers (Radcliffe and Ragsdale 2002). Infected mother plants cause secondary infections in their tuber progeny as virus particles are transported along with metabolites through the stolon into the tubers (Dupuis 2017; Malnoe et al 1994). It is an important issue for seed potato growers all over the world as vegetative propagation favours secondary infections in the field

Methods

Results

Discussion

Conclusion