Molecular quantification for differentiation of fresh and dried Jinqian Baihua She

Abstract

Freshly-used crude drugs have unique functions and advantages in TCM practice of treating diseases. Jinlong Capsule is a patent traditional Chinese medicine product effective for treatment of hepatocarcinoma, and fresh Jinqian Baihua She (JBS, the body of juvenile Bungarus multicinctus) is one of its important ingredients. The emergence of counterfeit fresh JBS, often identified as dried JBS with almost identical appearance, poses a difficult problem in the quality control of the product. Herein we report a molecular quantification-based method for differentiation of fresh and dried JBS by determining the copy number of a specific DNA marker in the samples. Using species-specific primers and TaqMan probes, we established a real-time quantitative PCR system for amplification of a fragment in the 658-bp cytochrome oxidase subunit I (COI) region from JBS specimens. The amplicon copy number in the muscle tissues ranged from 1.14 × 10⁷ to 4.83 × 10⁷ copies/mg in fresh JBS samples, as compared with 1.13 × 10⁵-8.91 × 10⁶ copies/mg in dried JBS samples. Based upon Fisher discriminant analysis, we used 1.27 × 10⁷ copies/mg as the cut-off value for differentiating fresh and dried JBS, which was validated in the single-blinded validation test of fresh and dried JBS samples. This qPCR system may provide an efficient means for accurate identification of fresh JBS to improve the quality control of the medicinal product.

Keywords: Freshly-used crude drugs; Jinqian Baihua She; Molecular quantification; QPCR.