Detection and absolute quantitation of Broad bean wilt virus 1 (BBWV-1) and BBWV-2 by real time RT-PCR.

Broad bean wilt virus 1 (BBWV-1) and BBWV-2 are the two most significant viruses in the genus Fabavirus, causing damage to many economically important agricultural crops worldwide. A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using two TaqMan(®)MGB probes was developed for sensitive and specific detection and quantitation of BBWV-1 and BBWV-2. Primers and probes were designed from conserved sequence stretches to detect all isolates of each virus. Standard curves using RNA transcripts identical to both TaqMan(®)MGB probes enabled absolute quantitation, with a wide dynamic range and high sensitivity (10(3)-10(10) RNA molecules). RT-qPCR was assayed with genetically divergent BBWV-1 and BBWV-2 isolates from different plant hosts and countries, and was used to evaluate the temporal accumulation of BBWV-1 RNA in two plant hosts.

Detection, discrimination and absolute quantitation of Tomato spotted wilt virus isolates using real time RT-PCR with TaqMan(®)MGB probes.

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan(®)MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidentalis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.

Discrimination between mild and severe Citrus tristeza virus isolates with a rapid and highly specific real-time reverse transcription-polymerase chain reaction method using TaqMan LNA probes.

Severe isolates of Citrus tristeza virus (CTV) inducing seedling yellows (SY) and/or stem pitting (SP) in grapefruit or sweet orange are a major threat for the citrus industry worldwide. Identification of these CTV variants was achieved by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) using a general primer set and three TaqMan locked nucleic acids (LNA) probes targeting sequences characteristic of severe, mild (non-SY, non-SP), and T36-like isolates. Successful amplification was achieved from fresh or silica-desiccated CTV-infected samples and all isolates but one reacted with one or more probes. Standard curves using RNA transcripts homologous to the three probes allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) copies. RT-PCR assays with homologous and heterologous transcript RNA mixes demonstrated that each probe reacted only with its cognate sequence which was detected even at ratios below 2.5%. Analysis of 56 pathogenically distinct CTV isolates from 20 countries showed that mild isolates reacted only with the mild probe, whereas severe SP and SY isolates reacted with the severe-SP or the T36-like probes, respectively, and often with a second probe. This procedure can be useful to identify and control potentially dangerous CTV isolates in areas affected only by mild isolates.

[Lymphedema as an inusual presentation of prostate carcinoma]. / Linfedema como presentación inusual de cáncer de próstata.

The most common etiology of lymphedema in our environment is tumoral and, generally, it is due to treatment of the cancer itself. However, the initial presentation of prostate carcinoma as lymphedema is very rare. In fact, there aren't very few documented cases in literature. We described the case of a patient who was admitted into the hospital to be examined for lymphedema in his genitals and legs. Our patient was diagnosed of prostate carcinoma with retroperitoneal adenopathies. The best treatment in this case is block-hormonal therapy. Prognosis is very bad and the survival rate is poor within five years of diagnosis.

Linfedema como presentación inusual de cáncer de próstata / Lymphedema as an inusual presentation of prostate carcinoma

La etiología más frecuente del linfedema en nuestro medio es la tumoral y generalmente debido al tratamiento del mismo. Sin embargo la presentación inicial del cáncer de próstata como linfedema es muy poco frecuente, habiendo muy pocos casos descritos en la literatura. Exponemos el caso de un paciente que ingresó para estudio de linfedema en región genital y en extremidades inferiores siendo diagnosticado de cáncer de próstata con adenopatías retroperitoneales. El tratamiento de elección es el bloqueo hormonal, siendo el pronóstico muy pobre y la supervivencia escasa a los cinco años del diagnóstico

The most common etiology of lymphedema in our environment is tumoral and, generally, it is due to treatment of the cancer itself. However, the initial presentation of prostate carcinoma as lymphedema is very rare. In fact, there aren’t very few documented cases in literature. We described the case of a patient who was admitted into the hospital to be examined for lymphedema in his genitals and legs. Our patient was diagnosed of prostate carcinoma with retroperitoneal adenopathies. The best treatment in this case is block-hormonal therapy. Prognosis is very bad and the survival rate is poor within five years of diagnosis

Preferential accumulation of severe variants of Citrus tristeza virus in plants co-inoculated with mild and severe variants.

The viral population in sweet orange plants, either healthy or pre-inoculated with the asymptomatic isolate of Citrus tristeza virus (CTV) T32, and then graft- or aphid-inoculated with the stem-pitting isolate T318, was characterized with respect to symptom expression, reaction with monoclonal antibody MCA13, single-strand conformation polymorphism (SSCP) of genes p18 and p20, bi-directional RT-PCR, and dot-blot hybridisation. All plants inoculated with T318, with or without pre-inoculation, showed stem pitting, reacted with MCA13, had the SSCP profile characteristic of this isolate, and in bi-directional RT-PCR yielded a 450-bp DNA product associated with severe isolates, indicating that T32 afforded no protection against T318. The latter isolate had two main sequence variants, the minor one of which was indistinguishable from the main T32 sequence, and both were detected in most plants that were graft-inoculated with T318. However, the T32 variant was not detected in plants that were aphid-inoculated only with T318 and also showed stem pitting. This suggested an association of symptoms with the major T318 sequence and preferential transmission of this variant by aphids. The T318-specific variant accumulated more than the T32 variant in plants in which both were replicating, suggesting a higher fitness of the former. Our results clearly emphasize the potential threat of severe CTV variants in areas where mild isolates are presently predominant.

The complete nucleotide sequence of a severe stem pitting isolate of Citrus tristeza virus from Spain: comparison with isolates from different origins.

The genomic RNA of the severe stem pitting Citrus tristeza virus (CTV) isolate T318A from Spain (19252 nt) was completely sequenced. It showed strong sequence similarities with the severe isolates SY568 from California and NUagA from Japan, and distant relationships with mild non-stem pitting isolates T385 from Spain and T30 from Florida. Contrasting with other severe CTV isolates, T318A had a predominant sequence variant even in the highly variable 5'-terminal untranslated region, in which a unique sequence variant (type II) previously associated with severe stem pitting isolates was detected. The high homogeneity of the T318A population suggests that the sequence obtained is probably responsible for the symptoms induced and makes it a useful tool to delimit pathogenicity determinants.