Enzyme-Mediated Duplex Exponential Amplification: A New Platform for Rapid Screening of <i>Hylurgus ligniperda</i>

Abstract

As the world's second largest timber importer, wood demand in China has been growing extremely rapidly, leading to an increase of 163% from 2009 to 2018. The plant quarantine pest <i>H. ligniperda Fabricius, 1787</i> is an invasive species frequently intercepted at ports. <i>H. ligniperda</i> causes damage mainly to pine and spruce. To improve the efficiency of on-site inspection and the efficacy of early detection, tight quarantine in ports, time-effective identification, and a national surveillance program for high-risk invasive bark beetles are in urgent need. In this study, a simple, fast and accurate classification method for <i>H. ligniperda </i>is established based on the enzyme-mediated duplex exponential amplification (EmDEA) technique. Partial region from <i>inhibitor of apoptosis 2</i> (<I>IAP2)</I> gene was selected as the target and 6 primer/probe combinations were designed. Through selection, the combination of 3-HY-F3, 3-HY-R2 and 3-HY-RNA5 was chosen as the final primer-probe set, as it showed the lowest Ct with highest final fluorescence signal. Method validation and specificity test using 6 other beetle species living on coniferous wood showed that this method is result reliable and specific. Through parameter analysis with positive plasmid, the detection limit was calculated to be 13.6 copies/μL (9×10<sup>-7</sup> ng DNA/ reaction), much higher than conventional molecular methods such as PCR. The whole process including isothermal amplification, data analysis, and result output can be finished in 30 min, which is highly time-effective. Besides, the operation is simple and little training is needed for non-professionals. The application prospects of this rapid screening system include customs screening in ports, wild survey in non-lab situations and early warning system development. The new analysis platform EmDEA, can also be implemented in rapid detection and identification of other forestry pests.