Column on Quality Evaluation of Arnebiae Radix

The screening of identification primers for Arnebiae Radix based on nested PCR*

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  • 1. National Institutes for Food and Drug Control, Beijing 102629, China;
    2. Shenyang Pharmaceutical University, Shenyang 110016, China;
    3. Yili Institute of Inspection, Testing and Certification, Yili 835000, China

Received date: 2023-10-02

  Online published: 2024-06-20

Abstract

Objective: To design and screen specific primers for efficient amplification and identification of Arnebiae Radix from market based on the concept of nested PCR. Methods: Nested primers was designed using the software of Primer Premier 5 based on the ITS sequence of Arnebia euchroma and the ITS2 sequence of non-pharmacopoeial Arnebiae Radix. The amplification efficiency of genomic DNA by ITS2 universal primers PCR and nested PCR was compared. The genomic DNA of Arnebiae Radix was amplified directly by nested primers and was detected by agarose gel electrophoresis. The specific primers designed for Arnebiae Radix based on the fragment length and variation sites’ coverage of the amplified product was evaluated. Results: A total of 11 primers were selected for synthesis after the primers were designed by Primer Premier 5 software. The amplification efficiency of nested PCR was superior to ITS2 universal primers PCR in genomic DNA of Arnebiae Radix. The results of nested primers directly amplified genomic DNA of Arnebiae Radix by agarose gel electrophoresis were better than those of ITS2 primers, and showed a single band. Four pairs of primers, AE-9S/AE-2A, AE-4S/AE-10A, AE-12S/10A, AE-29S/AE-29A, were determined to be suitable for the identification of Arnebiae Radix. Conclusion: On the basis of DNA barcode identification and nested PCR technology, 4 pairs of specific primers are identified which can be used to effectively distinguish Arnebia euchroma from the mainstreamed non-pharmacopoeial Arnebiae Radix in the medicinal materials market, providing reference for the subsequent research and development of identification methods for Arnebiae Radix and other traditional Chinese medicines.

Cite this article

LIU Jie, GU Hai-yuan, DAI Sheng-yun, QIAO Fei, LIAN Chao-jie, ZHENG Jian, JIA Sha-er·SI Ha-ke . The screening of identification primers for Arnebiae Radix based on nested PCR*[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44(5) : 756 -765 . DOI: 10.16155/j.0254-1793.2024.05.03

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