2018 ASHS Annual Conference

When performing diagnostics on plant samples, it is desirable to use methods that have low inputs of time and money, while still being effective. This is especially true with RNA virus extractions, like ones done for the diagnosis of Rose rosette virus. The kits that are used in RNA extraction are typically costly and time consuming, with some kits taking as much as 2 hours per sample. From a diagnostic stand point, this is high time input, especially when other diagnostic procedures can be completed quickly. Whenever the need arose to extract 100+ samples at a time, the kit extraction methods were not feasible for use because of the costs and the time required. Recently a direct antigen extraction method was developed for the Rose rosette virus. However, this method is still time and resource consuming and not for practical use in a diagnostic lab. This cost effective direct antigen extraction method was modified to allow for rapid extraction (15 min per sample) and the use of common resources in diagnostic clinics such as mesh bags and phosphate-buffered saline/tween (PBST). Extracts using the modified direct antigen method and the Qiagen RNeasy Plant Mini Kit are similar in sensitivity. The extracts are stable for repeated use, however Ct values on Takara Reverse Transcription quantitative PCR (qPCR) tend to increase by 1 to 2 cycles after repeated freeze-thaw cycles on the extract. However, most diagnostic labs do not retain extracts for research, so this may not be a problem. This extraction method is also effective at extracting stable RNA with other rose RNA viruses that can be used for detection.