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

Abstract

A RT-PCR was developed for the simultaneous detection and identification of three groups of Pepino mosaic virus (PepMV): European/Peruvian, Chilean 1/US1 and Chilean 2/US2 groups, followed by a restriction analysis that allowed the separation of the European, Peruvian, Chilean 2 and US2 isolates (patent pending). The multiplex RT-PCR reaction was performed by a mix of six primers that amplified a part of the RNA-dependent RNA polymerase gene of PepMV plus an internal control. Amplifications resulted in a 980 bp, 703 bp or 549 bp PCR product for European/Peruvian, Chilean 1/US1 or Chilean 2/ US2 groups, respectively. For the identification of the isolates present within the European/Peruvian and Chilean 2/US2 groups, the amplified PCR fragments were directly digested with SacI enzyme. The multiplex RT-PCR method presented higher sensitivity to detect CH1/US1 isolates in field samples than the RFLP-PCR method described by Hanssen et al. (European Journal of Plant Pathology 121:131-146, 2008). The detection limit observed with the multiplex RT-PCR was equal to or 3,125 times higher when compared to single RT-PCR or ELISA-DAS and molecular hybridisation methods, respectively. The use of the multiplex RT-PCR method in routine analysis of field tomato samples allowed the detection of 36.2 and 33.4% more positives when compared to the serological and molecular hybridisation methods, respectively, and the identification of plants infected with one, two or three isolates of PepMV.

Pepino mosaic virus genotype shift in North America and development of a loop-mediated isothermal amplification for rapid genotype identification

“…Currently, several molecular-based methods have been developed to determine the genetic diversity of PepMV, including restriction fragment length polymorphism (RFLP) [34,35], reverse transcription- polymerase chain reaction (RT-PCR) and sequencing [14,17], real-time RT-PCR [36], and hybridization [30]. All such molecular methods required some specialized instruments or sophisticated laboratory conditions, making it difficult to apply for field analysis.…”

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

“…In preliminary assays, ToTV was frequently found in tomato samples together with Pepino mosaic virus (PepMV), which is widely distributed in the tomato production areas of the country Verbeek et al, 2007a;Alfaro-Fernández et al, 2009b …”

“…In order to identify the specific isolate of PepMV in the infected samples, a multiplex RT-PCR assay was performed as described by Alfaro-Fernández et al (2009b). Briefly, this method identifies which of the five currently described genotypes of PepMV is present in the infected sample: European (EU), Peruvian (PE), Chilean 2 (CH2), US2 and/or CH1/US1 (or four strains: US1/CH1, EU, PE and CH2 strains, this last grouping also US2 genotype; Ling, 2007).…”

“…Tomato chlorosis virus (ToCV, Louro et al, 2000) and Tomato infectious chlorosis virus (TICV, Vaira et al, 2002), and by the multiplex RT-PCR procedure for the simultaneous detection of PepMV isolates, as described by Alfaro-Fernández et al (2009b). PCR was performed to confirm possible…”

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

“…In order to identify the specific genotype of PepMV in the infected samples, a multiplex RT-PCR assay was performed as described by Alfaro-Fernández et al (2009). Briefly, this method identifies the PepMV genotype present in the infected sample among the five determined genotypes.…”