Detection of G.elata using Enzymatic Recombinase Isothermal Amplification and Immunochromatography

• Published in: Analytical biochemistry 2024-01
• Main Authors: Ma, Qiuhe, Li, Tao, Liu, Yue, Chai, Jinjun, Xu, Ziqiang, Liu, Ang, Ma, Yuhe, Li, Mingcheng, Qu, Yongmei, Gao, Lijun
• Format: Article
• Language: English
• Subjects: compliance; Gastrodia elata; genome; health services; immunoaffinity chromatography; Oriental traditional medicine; polymerase chain reaction; quality control; recombinases; species
• ISSN: 0003-2697
• DOI: 10.1016/j.ab.2024.115618

The objective of this research is to develop two methodologies, Enzymatic recombinase amplification (ERA) and Polymerase Chain Reaction (PCR) coupled with Lateral Flow Dipstick (LFD), for the swift authentication of Gastrodia elata. Primers and nfo probes for the ERA of Gastrodia elata were developed based on the ITS2 genome sequences of Gastrodia elata and its counterfeits. Specific primers for the PCR analysis of Gastrodia elata were generated using the NCBI (National Center for Biotechnology Information) online platform. Through experimental validation, the optimal reaction system and conditions for both methodologies were established, and their efficacy was assessed. The methodologies developed herein are applicable for the targeted analysis of the medicinal species, Gastrodia elata. The sensitivity of the ERA-LFD detection method matched that of the conventional PCR-LFD approach, recorded at 1 ng·μL⁻¹. Consistency was observed in the results across three replicates of visualization test strips for both techniques. Upon evaluation, both the PCR-LFD and ERA-LFD methods demonstrated a total compliance rate of 100%. The ERA-LFD and PCR-LFD methods facilitate reduced detection times and offer visual results. These techniques are particularly effective for on-site detection and quality control in the authentication of Gastrodia elata within traditional Chinese medicine markets and at the primary level of healthcare provision.