T/SAIA 001-2021 DNA Molecular Identification Procedures for Bupleurum Seeds

Scope

Scope: This document specifies the principles, methods, and operating procedures for DNA molecular identification of Bupleurum seeds using real-time fluorescence PCR technology. This document is applicable to authenticating the seeds of traditional Chinese medicinal materials, including North Bupleurum and Narrow-leaved Bupleurum. Main technical content: Based on real-time fluorescence PCR technology, a multiple real-time fluorescence PCR detection system was established. The ITS gene sequence in DNA barcoding was used as the target gene, and universal primers and probes for Bupleurum were designed, along with specific primers and probes for Narrow-leaved Bupleurum and North Bupleurum. Through optimization and screening of the fluorescent polymerase chain reaction (PCR) reaction system and reaction conditions, a multiple real-time fluorescence PCR method was established, which can simultaneously identify the source components of Narrow-leaved Bupleurum and North Bupleurum seeds in the same PCR reaction system. DNA extraction: For each sample, 0.3g was mixed and ground into powder using a sterilized mortar under liquid nitrogen. The CTAB extraction buffer containing 2% beta-mercaptoethanol was preheated in a 65℃ water bath. Take 0.15g of the ground sample and place it in a 2.0mL centrifuge tube. Add 700μL of the 65℃ preheated CTAB extraction buffer and incubate at 65℃ for 30 minutes, shaking 3-4 times during this period. Add 700μL of chloroform/isopropanol (24:1), gently invert to mix thoroughly, and centrifuge at 12,000r/min for 10 minutes to extract the supernatant. Transfer the supernatant to another centrifuge tube, add 2/3 of the volume of -20℃ pre-cooled isopropanol, mix thoroughly, and incubate at -20℃ for 30 minutes or overnight. Centrifuge at 12,000r/min for 15 minutes to collect the precipitate. Add 700μL of 70% anhydrous ethanol to rinse, centrifuge at 12,000r/min for 2 minutes, and discard the supernatant. Repeat this step once. Dry the DNA naturally at r

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