Detection and relative quantitation of Soil-borne cereal mosaic virus (SBCMV) and Polymyxa graminis in winter wheat using real-time PCR (TaqMan®)

Soil-borne cereal mosaic virus (SBCMV) was first reported affecting wheat crops in Italy in 1960 and has spread subsequently to many other European countries, including the UK. SBCMV causes a serious disease of wheat, reducing yield by up to 70%; growing resistant varieties represents the only economical means of control. Real-time RT-PCR and PCR assays based on TaqMan(R) chemistry were developed for the detection and quantitation of SBCMV and its vector, Polymyxa graminis. Each assay incorporated an RNA or DNA specific internal control to facilitate quantitation. Nucleic acid extracts from SBCMV-infected plants were diluted in a nucleic acid extract from a healthy plant and amplified by real-time PCR to produce a standard curve. The standard curve was used to quantify the amount of SBCMV and P. graminis in plant samples. The sensitivity of the real-time assays were compared to established serological quantitation and conventional PCR methods by testing a range of SBCMV-infected wheat varieties. The results indicate that real-time assays were a 1000 times more sensitive than ELISA for the quantitation of SBCMV, and a 100 times more sensitive than conventional PCR for the quantitation of P. graminis. Real-time assays enabled sensitive, reproducible and specific detection of both virus and vector in wheat tissues. The real-time assays are potentially useful tools for determining variations in virus and vector concentrations in plant tissue from wheat varieties differing in resistance to SBCMV. Crown Copyright (C) 2004 Published by Elsevier B.V. All rights reserved.