Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

Abstract

28Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important 29 viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam 30 production globally. 31Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential 32 component of disease control. Current serological and PCR-based diagnostic methods for YMV are 33 time consuming involving a succession of target detection steps. 34In this study, a novel assay for specific YMV detection is described that is based on isothermal 35 reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown 36 to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-37 infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain 38 reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages 39 over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only 40 requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay 41 a promising candidate for adapting into a field test format to be used by yam breeding programmes or 42 certification laboratories.

An isothermal based recombinase polymerase amplification assay for rapid, sensitive and robust indexing of citrus yellow mosaic virus

“…The overall carrying cost of the experiment is less than carrying out PCR based thermal amplification (involves use of commercial kit for nucleic acid extraction, thermal cycler, and reagents). RPA has significantly reduced man-power and time in comparison with PCR based methods (Silva et al, 2015). The rapid kinetics and its simplicity attributes of RPA assay makes it as an ideal technique and more advantageous over other molecular diagnostic tools.…”

“…LAMP also had issues in regarding yielding reliable results as it generates multimeric products (Bakheit et al, 2008). As an alternate to the techniques available, a simplified isothermal based amplification technique, recombinase polymerase amplification (RPA) has been recently developed and successfully employed in detecting a few plant viruses from the crude sap (Kapoor et al, 2017;Londono et al, 2016;Mekuria et al, 2014;Silva et al, 2015;Zhang et al, 2014). RPA assay can be carried out at 37°C in a dry bath or an incubator and there is no requirement of expensive thermal cycler.…”

Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria

“…These advantages make RPA particularly suitable for use in remote regions (Supplemental Table S1; https://doi.org/10.3168/jds.2017-12566). Currently, the cost of the RPA-LF assay is higher than that of PCR-LF; however, this cost is expected to decrease with increased development and usage (Silva et al, 2015). The LF strips are promising tools that translate isothermal amplification into a visual test and eliminate the need for trained personnel, expensive materials, and other external apparatus.…”

Evaluación de la técnica de amplificación por recombinasa y polimerasa (RPA) para la detección de begomovirus presentes en cultivos de soja y poroto en Argentina

“…En la RPA, la amplificación de fragmentos específicos de ADN se logra a través de una combinación de proteínas (gp32, usvX y usvY) y la enzima Bacillus subtilis DNA polimerasa I, en presencia de ATP y un agente quelante (generalmente polietilenglicol de alto peso molecular) (Lobato y O'Sullivan, 2018; Valasevich y Schneider, 2017). El producto de RPA puede ser visualizado en geles de agarosa después de la purificación o desnaturalización de las proteínas involucradas en el proceso, aunque también existen métodos alternativos como fluorescencia y/o hibridación (Mekuria, Zhang y Eastwell, 2014;Silva, Bömer, Nkere, Lava Kumar y Seal, 2015;Zhang et al, 2014). Este método tiene una serie de ventajas con respecto a los mencionados anteriormente.…”