Visualized Nucleic Acid Hybridization Lateral Flow Strip Integrating with Microneedle for the Point-of-Care Authentication of Ophiocordyceps sinensis

Due to the price and demand of <i>Ophiocordyceps sinensis</i> having increased dramatically, adulteration with other fungi is a common problem. Thus, a reliable method of authentic <i>O. sinensis</i> identification is essential. In the present work, a rapid DNA extraction and double-tailed recombinase polymerase amplification (RPA) coupled with nucleic acid hybridization lateral flow strip (NAH-LFS) was developed to distinguish authentic <i>O. sinensis</i> ingredients from other fungi substitutes. In the presence of <i>O. sinensis</i>, the RPA amplicons with two ssDNA tails in the opposite ends, which could simultaneously bind with the SH-probes on gold nanoparticles (AuNPs) and capture the probe on the test line, formed visible red bands. RPA combined with NAH-LFS can efficiently detect <i>O. sinensis</i> DNA down to 1.4 ng/μL; meanwhile, the specificity test validated no cross reaction with common adulterants, including <i>Cordyceps gunnii</i>, <i>Cordyceps cicadae</i>, <i>Cordyceps militaris</i>, <i>yungui Cordyceps</i>, and <i>Ophiocordyceps nutans</i>. The whole RPA-NAH-LFS could be completed within 16 min. The RPA-NAH-LFS results in detecting 20 commercial <i>O. sinensis</i> samples are consistent with PCR-AGE and RT-PCR, confirming the feasibility of the RPA-NAH-LFS method. In conclusion, these results are expected to facilitate the application of RPA-NAH-LFS in the authentication detection of <i>O. sinensis</i> materials, providing a convenient and efficient method for <i>O. sinensis</i> quality control.