Métodos de RT-PCR e de hibridização Dot Blot para detecção universal de tospovirus

Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.Visando a um método para a detecção universal de tospovírus, utilizaram-se as técnicas de "Transcriptase reverse - polymerase chain reaction" (RT-PCR) e hibridização com sondas marcadas com digoxigenina. As espécies de tospovirus testadas foram: Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Os oligonucleotídeos foram sintetizados para anelar em regiões conservadas do genoma viral, sendo os produtos de PCR utilizados como sondas para hibridização através de "dot blot". Através de RT-PCR, utilizando-se diferentes combinações de oligonucleotídeos, somente foi possível a amplificação de todas as espécies quando se utilizou RNA de vírus purificado, sendo que, para a detecção a partir de RNA total, a RT-PCR apresentou problemas para a detecção das espécies ZLCV e IYSV. Sob condições de baixa adstringência, os testes de hibridização por "dot blot" com a sonda M (correspondente ao gene G1/G2) detectaram todas as espécies testadas, exceto IYSV. Com a sonda L, também sob condições de baixa adstringência, pôde-se detectar todas as espécies de tospovirus testadas simultaneamente em um único ensaio. Este método de detecção pode ser utilizado em serviços de quarentena e para indexação de germoplasma in vitro

The validity and reliability of the Portuguese versions of three tools used to diagnose delirium in critically ill patients

OBJECTIVES: The objectives of this study are to compare the sensitivity and specificity of three diagnostic tools for delirium (the Intensive Care Delirium Screening Checklist, the Confusion Assessment Method for Intensive Care Units and the Confusion Assessment Method for Intensive Care Units Flowsheet) in a mixed population of critically ill patients, and to validate the Brazilian Portuguese Confusion Assessment Method for Intensive Care Units. METHODS: The study was conducted in four intensive care units in Brazil. Patients were screened for delirium by a psychiatrist or neurologist using the Diagnostic and Statistical Manual of Mental Disorders. Patients were subsequently screened by an intensivist using Portuguese translations of the three tools. RESULTS: One hundred and nineteen patients were evaluated and 38.6% were diagnosed with delirium by the reference rater. The Confusion Assessment Method for Intensive Care Units had a sensitivity of 72.5% and a specificity of 96.2%; the Confusion Assessment Method for Intensive Care Units Flowsheet had a sensitivity of 72.5% and a specificity of 96.2%; the Intensive Care Delirium Screening Checklist had a sensitivity of 96.0% and a specificity of 72.4%. There was strong agreement between the Confusion Assessment Method for Intensive Care Units and the Confusion Assessment Method for Intensive Care Units Flowsheet (kappa coefficient = 0.96) CONCLUSION: All three instruments are effective diagnostic tools in critically ill intensive care unit patients. In addition, the Brazilian Portuguese version of the Confusion Assessment Method for Intensive Care Units is a valid and reliable instrument for the assessment of delirium among critically ill patients

Busca por Tomato yellow leaf curl virus e Tomato yellow leaf curl Sardinia virus em tomateiros Survey on Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus in tomatoes

A doença causada pelo complexo de vírus do tomato yellow leaf curl (TYLC) é muito séria em tomateiro, principalmente na América Central e Europa, e é causada por um complexo de begomovírus monopartidos. A doença torna-se predominante, mesmo em áreas com a presença de outros begomovírus. No Brasil, os problemas advindos da infecção por begomovírus uram entre os principais fatores de perdas e oneração de custos. A introdução do complexo TYLC representa uma grande ameaça para os produtores. Este estudo visou a realização de testes de detecção baseados em reação de polimerase em cadeia (PCR) e hibridização específicos em amostras suspeitas coletadas no estado de São Paulo. Um total de 46 amostras com sintomas lembrando aqueles causados pelo complexo TYLC foram coletados no município de Campinas. Todas as amostras foram negativas para detecção de Tomato yellow leaf curl virus e Tomato yellow leaf curl Sardinia virus, as duas espécies mais importantes do completo TYLC. Este resultado sugeriu que as duas espécies de vírus ainda não foram introduzidas no Brasil ou que ainda não apresentam uma larga distribuição.<br>The disease caused by the virus complex of tomato yellow leaf curl (TYLC) is extremely severe in tomato plants, notably in Central America and Europe, and is caused by a complex of begomovirus species. The disease becomes predominant even in areas with the presence of other begomoviruses. In Brazil. the problems arisen from begomovirus infection are one of the major factors for damages and increasing the costs. The introduction of the TYLC complex implies in a great threat for growers. This study aimed to carry out detection tests based on PCR (polymerase chain reaction) and specific hybridization on suspected samples collected in the São Paulo State, Brazil. A total of 46 samples with symptoms resembling those caused by the TYLC complex were collected in Campinas county. All samples were negative for the detection of Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus, the two most important virus from the TYLC complex. This result suggested that the two species have not yet been introduced in Brazil or that they are not widespread