[Development of a PCR method based on CYC2 for specific identification of Bauhinia species in edible flowers]

Bauhinia species are known for their medicinal and nutritional value in south China, with their flowers and tender leaves widely as important food materials and health food ingredients. However, their similar appearance to other plants, especially the toxic species, creates a serious risk of confusion with toxic plants during collection, processing, and marketing. A notable case is the edible B. acuminata, which closely resembles the highly toxic Rhododendron decorum. Multiple poisoning cases have resulted from this confusion in the marketplace, necessitating rapid and reliable identification methods. We screened the genetic sequences of Bauhinia and related plants from GenBank by comparing them systematically. Among all sequences tested, CYC2 showed the most distinct differences between target and non-target species, serving as a specific endogenous reference gene for Bauhinia. We then developed a rapid, specific, and feasible detection method by combining conventional PCR and real-time PCR. We designed and tested four sets of PCR primers and developed one TaqMan probe targeting CYC2. Testing on two target Bauhinia species (B. variegata and B. acuminata) and five non-target species showed that the optimized primers YTJ-3F/R produced the expected band at 147 bp only for the target species. No signal was detected in any non-target plants, including R. decorum. Conventional PCR achieved a limit of detection (LOD) of 0.02 ng DNA (0.01% adulteration rate, W/W), while the TaqMan assay reached a LOD of 0.4 ng (0.2% adulteration rate, W/W). The calibration curve was y=-3.346x+31.758 (R²=0.996), with the amplification efficiency of 99.0%. Practical applicability of the established method was evaluated through the analysis of 20 commercially available edible flower samples, and repeatability was confirmed over three consecutive days, which demonstrated stable and reproducible results across all replicates. This study provides a rapid and specific molecular detection approach for the authentication of Bauhinia-derived edible plant materials. At the methodological level, this method exhibits satisfactory sensitivity and specificity. The findings of this study offer a technically feasible pathway for adulteration detection and food safety risk management of edible flower raw materials, with practical implications for market regulation and the prevention of food safety incidents involving edible floral ingredients.