RNaseH依赖性PCR扩增结合熔解曲线法鉴别培植牛黄与体外培育牛黄OA

Objective To achieve rapid discrimination between cultivated bezoar and in vitro cultivated bezoar using ribonuclease H-dependent PCR amplification combined with melting curve analysis(rhPCR-HRM).Methods Specific rhPCR-HRM primers were designed based on sequence variations in the mitochondrial COI gene between bovine and porcine species.Discrimination was performed via rhPCR amplification by analyzing differences in the melting temperature(Tm)values of the amplification products.The specificity of the primers was evaluated,reaction conditions were optimized,and the sensitivity and practical applicability of the method were investigated.Results The designed primers demonstrated excellent specificity,successfully distinguishing bovine-derived and porcine-derived samples.The amplification products exhibited single,specific melting curve peaks with corresponding Tm values of(83.93±0.2)℃and(79.15±0.2)℃,respectively.Furthermore,the method showed high sensitivity,capable of detecting DNA templates as low as 0.05 ng.Testing of 44 market-collected medicinal material samples revealed that among 30 samples labeled as cultivated bezoar,only 7 matched the characteristics of cultivated bezoar,while the remaining 23 were actually in vitro cultivated bezoar.All 14 samples labeled as in vitro cultivated bezoar were confirmed.Conclusion The established rhPCR-HRM method effectively differentiates cultivated bezoar from in vitro cultivated bezoar,exhibiting high specificity and sensitivity.It provides a novel approach for authenticity identification of bezoar and offers robust technical support for market regulation.