Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of prunus necrotic ringspot virus in herbaceous and Prunus hosts

Abstract

Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and woody plants: DAS-ELISA, nonisotopic dot-blot hybridization and reverse transcriptional polymerase chain reaction (RT-PCR). When purified virus preparations were used, the detection limit of the RT-PCR technique was 1·28 pg mL ¹1 whereas nonisotopic molecular hybridization and DAS-ELISA allowed detection of 0·8 ng mL ¹1 and 4 ng mL ¹1 , respectively. Several sample processing procedures were evaluated for virus detection by the nonisotopic molecular hybridization technique. When a very short and simple sample processing method was used, the detection limit of the nonisotopic molecular hybridization technique was 25 times higher than that of DAS-ELISA and 625 times lower than that of RT-PCR. A comparison of the level of virus accumulation in mature fruits and in leaf tissue showed that, on average, 125 times more virus was found in the fruits. Materials and Methods Plant and virus sourcePetals from a field-grown PNRSV-infected nectarine (Prunus persica) tree were homogenized and served as inoculum for cucumber (Cucumis sativus cv. National Pickling) plants. After several passages, virus was Plant Pathology (1998) 47, 780-786 780 ᮊ 1998 BSPP

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

“…The obtained result of the higher sensibility of this molecular method compared to the ELISA is in accordance to those reported in previous assays for PepMV or other viruses (Sánchez-Navarro et al, 1998) vaporariorum (Wisler et al, 1998b;Pospieszny et al, 2007;Amari et al, 2008), and large populations of whiteflies often co-infect tomato fields worldwide. In addition since the first outbreak of ToCV in Spain, which affected tomato crops with an incidence of more than 30% of symptomatic plants in individual fields (Navas-Castillo et al, 2000), this virus has commonly infected tomato crops during different growing seasons (EPPO, 2005).…”

“…Although the multiplex RT-PCR procedure presented herein contains a cocktail of six different primers, no differences in terms of detection limit were observed when a serially-diluted double-infected sample was analyzed by either the multiplex (containing a cocktail of six primers) or the single (containing two primers) RT-PCR. When the similarly diluted samples were analyzed by ELISA or dot-blot hybridization, the detection limit of the multiplex reaction was 3125 (5 5 ) times higher, a difference that is in the range of previously reported results (Sánchez-Navarro et al, 1998;Saade et al, 2000).…”

“…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).…”

El virus del mosaico del pepino dulce (Pepino mosaic virus) afectando al cultivo del tomate (Solanum lycopersicum): Caracterización y Epidemiología

“…With all the surveyed samples, non isotopic dot-blot hybridisation was performed as previously described by Sánchez-Navarro et al (1998) in order to detect the Parietaria mottle virus (PMoV) using a dig-RNA probe complementary to a fragment of the coat protein of PMoV provided by Dr. Pallás (IBMCP-UPV, Valencia, Spain). One microlitre of total RNA was firstly denatured with formaldehyde and then directly applied onto a nylon membrane.…”

Epidemiología, caracterización molecular y desarrollo de métodos de diagnóstico del virus de las manchas necróticas del melón (MNSV) y de su hongo vector Olpidium Bornovanus.

“…Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) and immunosorbent electron microscopy (ISEM) have been used to detect MNSV. Molecular hybridization and reverse transcription-polymerase chain reaction (RT-PCR) have also been used to obtain increased sensitivity , as observed in other viral systems (Sánchez-Navarro et al, 1998).…”