Title : Recombinase polymerase amplification coupled with lateral flow tests for high sensitive detection of potato pathogens
Abstract:
Phytopathogenic bacteria, viruses and viroids are serious threats to agricultural and natural ecosystems. Rapid, accurate and sensitive screening tests are necessary to prevent worldwide spreading of plant diseases. Control of seed materials and field plantings requires methods for on-site detection of plant pathogens. Competitive alternatives to polymerase chain reaction (PCR) and PCR-derived techniques are isothermal amplification methods such as recombinase polymerase amplification (RPA). This method has two main advantages – fixed temperature and short duration. The reaction proceeds at 30-42 ?C, and its recommended time is about 20 min. Moreover, RPA may be applied for non-laboratory control of plant pathogens due to possibility of its coupling with lateral flow assay (LFA). The LFA as registration tool provides such advantages as low cost and rapidity (<10 min). Here, we present benefits of the coupled RPA-LFA assays for sensitive detection of three potato pathogens of different nature. 1) The developed assay for potato virus X (PVX) has the detection limit (DL) equal to 0.14 ng PVX per g of potato leaves and 30-min duration. The PVX assay is 260 times more sensitive than conventional LFA based on antibodies and demonstrates the same sensitivity as PCR. 2) Dickeya solani, one of the most significant bacterial pathogens, infects potato plants, resulting in severe economic damage. The developed RPA-LFA has DL equal to 14,000 colony-forming units per gram of potato tuber. This sensitivity corresponds to PCR, but the assay is implemented at a fixed temperature (39 ?C) over 30 min. 3) Potato spindle tuber viroid (PSTVd) is the most primitive potato pathogen that causes perceptible crop damage. Its RT-RPA-LFA allows detecting 106 copies of in vitro transcribed PSTVd RNA in reaction or up to 1:106 diluted extracts of infected plants. The proposed adjustments for PRA-LFA can be applied for ultrasensitive and rapid detection of other plant pathogens. The study was supported by the Russian Science Foundation (grant # 16-16-04108).
