Identification of Scolopendra Dispensing Granules by Allele-specific PCR
10.13422/j.cnki.syfjx.20231111
- VernacularTitle:蜈蚣配方颗粒的位点特异性PCR鉴别
- Author:
Yuansheng XU
1
;
Li HU
2
;
Chao JIANG
2
;
Yuyang ZHAO
2
;
Tianyun CHEN
2
;
Hui ZHANG
3
;
Hui TIAN
1
;
Yuan YUAN
2
Author Information
1. Guangxi University of Chinese Medicine， Nanning 530200， China
2. National Resource Center for Chinese Materia Medica， China Academy of Chinese Medical Sciences， Beijing 100700， China
3. China Resources Sanjiu Medical & Pharmaceutical Co. Ltd.， Shenzhen 518029， China
- Publication Type:Journal Article
- Keywords:
Scolopendra;
dispensing granules;
single nucleotide polymorphism （SNP） locus;
polymerase chain reaction （PCR）
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2024;30(4):48-54
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo establish an allele-specific polymerase chain reaction （PCR） method for identifying Scolopendra dispensing granules， so as to ensure the quality and therapeutic effects of Scolopendra and its preparations. MethodThe primer interval suitable for the PCR was selected based on the cytochrome c oxidase subunit 3（COX-3） gene sequence of Scolopendra， and the single nucleotide polymorphism （SNP） loci of Scolopendra and its adulterants were mined from the interval for the design of specific primers. The samples of Scolopendra and its adulterants were collected. The PCR system was established and optimized regarding the annealing temperature， cycles， Taq enzymes， DNA template amount， PCR instruments， and primer concentrations， and the specificity and applicability of this method were evaluated. ResultThe PCR system was composed of 12.5 μL 2×M5 PCR Mix， 0.4 μL forward primer （10 μmol·L-1）， 0.4 μL reverse primer （10 μmol·L-1）， 2.5 μL DNA template， and 9.2 μL sterile double distilled water. PCR parameters： Pre-denaturation at 94 ℃ for 3 min， 30 cycles （94 ℃ for 20 s， 62 ℃ for 20 s， 72 ℃ for 45 s）， and extension at 72 ℃ for 5 min. After PCR amplification with the system and parameters above， the electrophoresis revealed a bright band at about 135 bp for Scolopendra and no band for the adulterants. ConclusionThe established allele-specific PCR method can accurately identify the medicinal materials， decoction pieces， and standard decoction freeze-dried powder of Scolopendra， as well as the intermediates and final products of Scolopendra dispensing granules， which is of great significance for ensuring the quality and clinical efficacy of Scolopendra and its preparations.
