Simultaneous detection and identification of eight stone fruit viruses by one-step RT-PCR

Simultaneous detection and identification of Pepino mosaic virus (PepMV) isolates by multiplex one-step RT-PCR

“…Previous results obtained by the multiplex RT-PCR assay have shown that a cocktail of seven pairs of primers affect the detection limit (Sánchez-Navarro et al 2005). Although the multiplex RT-PCR-SacI procedure presented here contains a cocktail of six different primers, no differences in terms of detection limit were observed when a serially-diluted double-infected sample was analysed by either the multiplex (containing a cocktail of six primers) or the single (containing two primers) RT-PCR.…”

“…The reaction was carried out with a mixture of all the primers listed in Fig. 1 concentration of 0.25 pmol µl −1 and the primers of the internal control Rbcl gene (0.05 pmol µl −1 ) corresponding to partial sequence of the ribulose 1.5-biphosphate carboxylase chloroplast gene (Sánchez-Navarro et al 2005). The PCR programme consisted of an initial incubation at 50°C for 30 min followed by 2 min at 94°C and 40 cycles of 94ºC for 15 s, 50°C for 30 s and 68°C for 1 min.…”

Occurrence of Stone Fruit Viruses in Plum Orchards in Latvia

“…Despite wide use in routine and screening diagnosis, ELISA tests may fail due to low viral titre and the inhibitory effect of woody plant sap compounds (Kinard et al, 1996;MacKenzie et al, 1997). Therefore, PCR based techniques, which are more sensitive and accurate, can provide an alternative for virus detection in woody plants (Kinard et al, 1996;Sánchez-Navarro et al, 2005;López et al, 2008;Jaroðová et al, 2010;Çevik et al, 2011).…”

Epidemiología y variabilidad patogénica del virus del mosaico del pepino dulce (Pepino mosaic virus). Nuevas enfermedades asociadas a su presencia (torrao o cribado)

“…El método resultó ser altamente específico incluso en infecciones múltiples, presentando a su vez un límite de detección 3125 veces mayor que el ELISA o la hibridación molecular, valor que se encuentra dentro del rango descrito para estas técnicas (Saade et al, 2000;Sánchez-Navarro et al, 1998;Sánchez-Navarro et al, 2006). El análisis de muestras de campo reveló la presencia de un gran porcentaje de muestras con infecciones mixtas de aislados pertenecientes a los genotipos EU con CH2 o CH1, representativos de dos regiones geográficas diferentes (Murcia y las Islas Canarias, respectivamente).…”

“…However, so far there is only a methodology based on a single RT-PCR-RFLP assay adjusted for simultaneous detection and identification of three (Martínez-Culebras et al, 2002) or five (Hanssen et al, 2008; here after RT-PCR-Hanssen) PepMV strains. The development of multiplex RT-PCR has been used successfully for the routine diagnosis of plant viruses (see James et al, 2006 for review;Ferrer et al, 2007;Nie and Singh, 2000;Sánchez-Navarro et al, 2006;Uga and Tsuda, 2005). This diagnostic method allows the simultaneous detection and identification of different viruses with less time and cost waste.…”