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 ofOphiocordyceps sinensishaving increased dramatically, adulteration with other fungi is a common problem. Thus, a reliable method of authenticO. sinensisidentification 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 authenticO. sinensisingredients from other fungi substitutes. In the presence ofO. sinensis, 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 detectO. sinensisDNA down to 1.4 ng/μL; meanwhile, the specificity test validated no cross reaction with common adulterants, includingCordyceps gunnii,Cordyceps cicadae,Cordyceps militaris,yungui Cordyceps, andOphiocordyceps nutans. The whole RPA-NAH-LFS could be completed within 16 min. The RPA-NAH-LFS results in detecting 20 commercialO. sinensissamples 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 ofO. sinensismaterials, providing a convenient and efficient method forO. sinensisquality control.