Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses

Detection and identification of TMV infecting tomato under protected cultivation in Paraná State

“…There are reports of isolates capable to overcome the resistance of some tomato varieties expressing, for example, the resistance factor 'Tm-1' that provides resistance for ToMV, and the resistance factors 'Tm-2' and 'Tm-2 2 ' for TMV (Moreira et al, 2003). Additionally few indicator plants distinguish reliably between TMV and ToMV (Jacobi et al 1998). In this work, electron microscopy examinations of ultratin sections revealed rod shaped particles, and combined with host range, allowed to conclude that a Tobamovirus was associated with the diseased tomatoes from Sapopema (Table 1 and Fig.1).…”

“…The antiserum produced reacted to leaf extracts from symptomatic plants in homologous serological reactions and not with healthy plants. According to some authors, serological crossreaction between ToMV and TMV limits the efficiency of serological differentiation of these two viruses (Jacobi et al, 1998). In this regard, low cross-reaction was observed by Duarte et al, (2002) for a monoclonal antiserum that showed strong specificity for ToMV, but problems for raising this kind of antibodies limited their immediate use.…”

“…The cDNA was synthesized using reverse transcriptase (Gibco BRL) and buffers according to the manufacturer in a final volume of 30µl. From the total RNA extraction solution, 10 ml were taken and mixed with 2µl of 10 µM reverse primer TMV-2 (5'-GAA AGC GGA CAGAAA CCC GCT G-3' ) or 2µl of reverse primer ToMV-5 (5'-CTC CAT CGT TCA CAC TCG TTA CT-3') (Jacobi et al, 1998). Afterwards, the samples were incubated at 70 0 C for 10 min and quenched on ice for at least 1 min.…”

“…Reverse primer TMV2 and forward primer TMV1 (5'-GAC CTG ACA AAAATG GAG AAG ATC T-3') are expected to amplify a 422 bp PCR product within the viral movement protein gene of TMV, while primer ToMV-5 and forward primer ToMV-6 (5'-GAT CTG TCA AAG TCT GAG AAA CTT C-3') amplify a product of 508 bp within the same gene of ToMV (Jacobi et al, 1998). PCR reactions were performed in a 40µl volume, containing 10mM each of dATP, dCTP, dGTP and dTTP, 2µl of each primer (10 µM), 2.5 U of Taq DNA Polymerase (Gibco BRL), 4µl of 10X PCR buffer, 25mM MgCl, and 4µl of the first strand synthesis product.…”

Biological, Serological and Molecular Characterization of Papper MildMottle Virus Isolated from Weast Region of Kingdom of Saudi Arabia

“…The IC-PCR approach has chosen rather than RT-PCR test because of its high sensitivity due to the initial immunocapture enrichment step followed by PCR amplification, high specificity due to the combination of the virus specific antibody capture and primer specificity, and virus detection directly in crude plant extracts rather than in partially or highly purified nucleic acid preparations. Studies on the detection of economically important fruit viruses have documented the potential of IC-RT-PCR to overcome limitations for virus detection in crude tissue extracts of woody plants [10,31,55]. Direct detection sensitivity with ELISA in the presence of PMMoV was not possible as the ELISA assay could not reliably distinguish PMMoV.…”