RESUMO
The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen-alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.
Assuntos
Vírus do Mosaico da Alfafa/isolamento & purificação , Nicotiana/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Sondas de Oligonucleotídeos/química , Doenças das Plantas/virologia , Recombinases/metabolismo , Solanum tuberosum/virologia , Vírus do Mosaico da Alfafa/genética , Bioensaio , Recombinases/genética , Transcrição Reversa , Proteínas Virais/genéticaRESUMO
Determination of local necrotic lesions on primary leaves infected by alfalfa mosaic virus (AMV) revealed that hydrocarbons (HC) contamination of the substrate used for cultivation of French bean (Phaseolus vulgaris L., cv. Black Turtle Soup) caused a reduction of bean leaf area and an increase of plant sensitivity to AMV infection. On the other hand, superficial contamination of the leaves by crude oil caused an inhibition of lesion formation. Changes of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of extractable bean leaf proteins related to the cultivation substrate contamination by HC were also detected.