RESUMO
Babaco is a hybrid cultivar native to the Andean region of Ecuador and Colombia, commercially attractive for its fruit. Babaco production in Ecuador faces losses from plant pathogens like babaco mosaic virus (BabMV), an RNA virus that causes chlorosis, leaf mottling, and deformation. Phylogenetic studies link BabMV to papaya mosaic virus (PapMV), alternanthera mosaic virus, and senna mosaic virus. To address this threat, we developed novel species-specific primers to detect BabMV targeting a 165 bp region of the coat protein (CP). Genus-specific primers were designed to validate the species-specific primers and attest their ability to discriminate between BabMV and its closest relatives. These primers targeted a 175 bp fragment of the CP region. The most effective sets of primers were chosen for reverse transcription polymerase chain reaction (RT-PCR) and SYBR® Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) in symptomatic and asymptomatic babaco plants. Among 28 plants tested, 25 were positive and 3 were negative for BabMV using species-specific and genus-specific primers in RT-PCR and RT-qPCR, while the PapMV positive control was detected with the genus-specific primers and was negative for the species-specific primers. These primers represent a valuable molecular tool for detecting BabMV, potentially enhancing crop management.
Assuntos
Primers do DNA , Doenças das Plantas , Doenças das Plantas/virologia , Primers do DNA/genética , Equador , Proteínas do Capsídeo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Especificidade da Espécie , ColômbiaRESUMO
Paper-based analytics allows building portable and disposable devices for point-of-care (POC) diagnosis. Conventional methods for quantifying proteins exhibit substantial disadvantages related to costs and difficulty of the technique when used in settings where fast and cost-effective assays are needed. We report the successful application of a simple, rapid, easy to use, and label-free aptasensor strategy based on the selective fluorescence of the NMM IX dye. For the probe design, the three-dimensional (3-D) structures of the DNA components were carefully analyzed using software for the 3-D visualization of crystallographic structures. The chimeric aptafluorescence molecule consists of two modules, a detection aptamer and a transduction sequence that induces the specific fluorescence of NMM IX. In the presence of thrombin, a fluorescent spot visible to the naked eye can be observed. The fluorescent response is directly proportional to protein concentration and can be easily quantified colorimetrically using a low-cost microscopy system. The recognition probe design might be adaptable to other relevant biological analytes by changing the sequence of the aptamer. This proof of principle represents a contribution to the development of useful, cheap, reliable, and simple protein quantification assays for POC testing.